WB: anti-flag (Orai1) WB: anti-stim1

Transcription

1 [Ca 2+ ] i (µm) Ca Ca 2 mm Ca 1 µm TG HeLa µm 2-APB HeLa O+S KDa DSP (mm) Complex Monomer KDa 2 1 DSP (mm) Complex Monomer (Orai1) WB: anti-stim1 Figure S1 Measurement of store-operated Ca 2+ entry (SOCE) in HeLa O+S cells and detection ti of high MW protein complexes containing Orai1 and STIM1 y DSP cross-linking. (a) Control HeLa cells or HeLa cells staly expressing Orai1 and STIM1 (HeLa O+S) were examined for SOCE. Inhiition of CRAC channel activity was monitored using 2-APB ( µm). Each trace represents average ± s.e.m from -6 cells. () HeLa O+S cells were treated with different concentrations of DSP for one hour. Cell lysates were cleared y ultracentrifugation and analyzed y non-reducing SDS-PAGE and immunolotted with anti-flag (Orai1, left) or anti-stim1 (right) antiodies.

2 Human MAAPDGRVVSRPQRLGQGSGQGPKGSGACLHPLDSLEQKETQEQTSGQLVMLRKAQEFFQ 6 Chimpanzee MAAPDGRVVSRPQRLGQGSGQGPKGSGACLHPLDSLEQKETQEQTSGQLVMLRKAQEFFQ 6 Mouse MATPSGREDSSSQ----TPGHGKQGSGACVEQLDHPEKLEVEMPD--QSAMWKKAQEFFQ 54 Zera fish MASFTSTCGSKVD RGSHHRGDSKGTPSESD CSAVIEKTREFFQ 43 EF hand motif 2 ** ** * EF hand motif 1 Tropomyosin domain Human TCDAEGKGFIARKDMQRLHKELPLSLEELEDVFDALDADGNGYLTPQEFTTGFSHFFFSQ ** ** * 12 Chimpanzee TCDAEGKGFIARKDMQRLHKELPLSLEELEDVFDALDADGNGYLTPQEFTTGFSHFFFSQ 12 Mouse TCDSEGKGFIARTDMQRLHQELPLSLEELEDVFDALDADGNGFLTPEEFTTGFSHFFFSQ 114 Zera fish ICDVEGKGFITRRDMQRLNGELPLSADDLENVFDSLDADANGYLTFEEFSSGFSEFMFGP 13 Human NNPSQEDVG--EQVAQRHEEKVYLSRGDEDLGDMGKDEEAQFRMLMDRLGAQKVLEDESD 178 Chimpanzee NNPSQEDAG--EQVAQRHEEKVYLSRGDEDLGDMGEDEEAQFRMLMDRLGAQKVLEDESD 178 Mouse NIQGEEEAD--QQVAQLQEEKVYQSRGEEDVGDMDHDEEAQFQMLMDRLGAQKVLEDESD 172 Zera fish SVVPADPHGGEELVSRKSPEMLYESQWEERLSRGEDDEEKHFCMLMENLGASNIFEDPEE 163 Coiled-coil domain Human VKQLWLQLKKEEPHLLSNFEDFLTRIISQLQEAHEEKNELECALKRKIAAYDEEIQHLYE 238 Chimpanzee VKQLWLQLKKEEPHLLSNFEDFLTRIISQLQEAHEEKNELECALKRKIAAYDEEIQHLYE 238 Mouse VRQLWLQLRKDEPHLLSNFEDLLTTIFAQLQEAHEQKNELECALRKKIAAYDEEIQHLYE 232 Zera fish VRSLWVQLRRDEPHLLSNFEEFLARVTYQIKEANQEKKEMETALKRKSATHDDEIQRLYE 223 Tropomyosin domain Human EMEQQIKSEKEQFLLKDTERFQARSQELEQKLLCKEQELEQLTQKQKRLEGQCTALHHDK 298 Chimpanzee EMEQQIKSEKEQFLLKVTLPSVSRGQDWRNPLLCQGKESECFSRKSGRLEGQCTALHHDK 298 Mouse EMEQQIKSEREQFLLKDTERFQARSRELEKKLSAKEQELERLNQKQ-RKVGYCGDIVG Zera fish EMEQQIKNEKDRILLEDSERFLTRSQDMEHQLLSKEKELEILSNKQKRLERQCRDLLSEQ 283 Tropomyosin domain Human HETKAENTKLKLTNQELARELERTSWELQDAQQQLESLQQEACKLHQEKEMEVYRVTESL 358 Chimpanzee HETKAENTKLKLTNQELARELERTSWELQDAQQQLESLQQEACKLHQEKEM Mouse ----PQLFQLSLP---LPHALHHSSMDF Zera fish RETSVENVKLKRYNEDLSRELDHTSQELSLAQEQLMLLQEQSSRLHEEREM Human QREKAGLLKQLDFLRCVGGHWPVLRAPPRSLGSEGPV 395 Chimpanzee Mouse Zera fish Human NP_ Chimpanzee XP_ Mouse NP_ Zera fish XP_ Figure S2 Amino acid sequence alignment of CRACR2A from various species. Amino acid sequences of human, chimpanzee, mouse and zerafish CRACR2A proteins were aligned using ClustalW2 (default options). The predicated conserved EF-hands are colored in light lue (predicted using SMART). The predicted coiledcoil sequence (Human Protein Reference Dataase and COILS) in the C terminus is oxed in gray and tropomyosin domains predicted using PRINTS are highlighted in lue. The CRACR2A peptide detected from mass spectrometry is shown in red. This peptide was detected in a gel fragment corresponding to MW of ~45 as descried in Fig. 1. In humans, three alternative transcripts of CRACR2A (NM_ , NM_ and NM_3268.3) were identified. Proteins derived from three isoforms are identical in their first 2 amino acids with differences in their C-terminal sequences. Amino acids sequences corresponding to isoform c of CRACR2A (predicted MW; 45.6, Compute pi/mw tool) have een aligned here.

3 Digitonin ProteinaseK GFP STIM1-YFP % Fluorescence orescence Orai1-YFP STIM1-YFP GFP GFP GFP-CRACR2A CRACR2A-GFP GFP-R2A % Flu Time (s) R2A-GFP % Fluoresc cence After Digitonin Pre-treatment Digitonin ProteinaseK Figure S3 CRACR2A is a cytosolic protein as judged d y fluorescence protease protection ti (FPP) assay. (a) HEK293 cells expressing either N- or C-terminally GFP-tagged CRACR2A proteins were treated with digitonin to permeailize the cells and allow for diffusion of cytoplasmic proteins outside the cell. Susequent treatment of cells with proteinase K leads to degradation of GFP tags exposed to the cytoplasm and therey reduction in fluorescence. STIM1-YFP and Orai1- YFP used as controls for proteins integral to the ER and plasma memrane respectively, showed reduction in fluorescence upon proteinase K treatment (top graph) due to cytoplasmic localization of their YFP tags. Bar, 2 μm () Graphical representation of the images shown in panel a. The initial intensity of fluorescence was set to %. Each trace represents average ± s.e.m from 3- individual cells. The ar graph shows average ± s.e.m from three different experiments.

4 M M 1 1 : ng Antiody Production 2 Purified His- CRACR2A c His-CRACR2A Purification WB: Anti-CRACR2 Vec R2A R2B Vec R2A R2B Vec R2A R2B 15 d GST-Orai1 fragments purification anti-cracr2a anti-actin His-CRACR2A fragments purification 2 15 M M 2 GST Orai1-N Orai1-IC Orai1-C ΔN ΔC EF2MUT Figure S4 Purification of recominant CRACR2A and dorai1 proteins and generation of anti-cracr2a ticracr2a antiodies. (a) E. coli expressing 6X His-tagged CRACR2A proteins were either left uninduced or induced with.2 mm IPTG, and lysates were analyzed for CRACR2A expression. Eluates show purified CRACR2A protein (red ox). The purified protein ands were excised to generate anti- CRACR2A antiody. Proteins were detected y coomassie lue staining. () Polyclonal antiody against CRACR2A generated in raits was examined for sensitivity to detect purified CRACR2A protein.(c) Examination of specificity of CRACR2A antiody. Lysates from HEK293 cells expressing either empty vector (Vec), CRACR2A, or CRACR2B were examined y immunolotting with anti-flag antiody to detect oth proteins (left), with CRACR2A antiody that t detects t only CRACR2A protein (middle panel), and with anti-actin antiody (right panel) as a loading control. (d) Lysates of E.coli expressing either GST or the N terminus (Orai1-N), the intracellular loop (Orai1-IC) or the C- terminus (Orai1-C) of Orai1 were separated y SDS- PAGE (laeled as induced) together with purified fragments (laeled as purified) and visualized y coomassie lue staining (left). Lysates of Ecoli E.coli expressing 6X His-tagged truncation or point mutants of CRACR2A proteins were analyzed y SDS-PAGE and coomassie lue staining (right). Uninduced, induced cell lysates and proteins purified using Ni 2+ resins were examined. M, molecular weight marker.

5 hcracr2b transcript (Enseml Transcript ID ENST4448, NM_173584) hcracr2b Expected transcript (Enseml Transcript ID ENST327417) hcracr2b transcript (sequenced)? mcracr2b transcript (NM_113.2) c atggccagccctggaaagcctggggctgatgaggcccaggaggaggagggggaactcgaggggggctctgcagggccgcgggctgcaatactggagcaggctgaggagctgtttctgctgtgtgacaaggaggcta agggcttcatcaccaagcacgacctgcagggtctccagagcgacctgcccctcacgccagagcagctggaggctgtgtttgaaagtctggaccgggctcacactggcttcctcaccgccagggagttctgcctgggcctgg ggatgtttgtgggggtggcgtcagcccagggagcgaacccctgcaggactcccgaggagacctttgagtcgggcgggctcgacgtgcagggcacggcgggctctctggatgaggaggaggaagaggaggagcgattc cacactgtgctggagcagctgggggtggccccggtcctgggcaagcagcgggctgtgaggacgctctgggccaggctgcagcgcgagcgccccgagctgctgggctctttcgaggatgttctgatacgcgcgtcggcctg cctggaggaggcggcccgggagcgcgacggcctggagcaggcgctgcggaggcgcgagagcgagcacgagagggaggtgcgcgctctgtacgaggagacggagcagcttcgggagcagagccggcgcccgc cgagtcagaacttcgcccgcggggagcggagaagccgtctggagctggagctgcagagccgcgagcaggacctggaacgcgcgggcctgcggcagcgggagttagagcagcagctgcacgcccaggctgcgga gcacctggaggcacaggcccagaactcccagctgtggcgggcgcacgaggcgctgcgaacgcagctggagggggcgcaggagcagatccgcaggctggagagcgaagcacgaggccgccaggagcaaaccc aacgagacgtggtcgccgtctccaggaacatgcagaaagagaaagtcagcctgctacggcaactggagctgctcagggagctgaatacacggctgcgggatgacagggacgcctgcgaggccaggcgggcgggca gcagctgcaggaaggctctgacaacagcccgcctgcctgggcccacctgctgctgctgctgttgctgggctcggccccccagacgcggctctggccaccttcccagtgcccggtga hcracr2b sequenced clone mcracr2b NP_12274 EF hand hcracr2b MASPGKPGADEAQEEEGELEGGSAGPRAAILEQAEELFLLCDKEAKGFITKHDLQGLQSD 6 mcracr2b MASPGLPGSGEGQEGE-ETTGVSARHGVEVLQQAQELFLLCDKDAKGFITRQDLQGLQSD 59 ***** **:.*.** * * * **. :*:**:********:******::******** 76EF hand 2 87 hcracr2b LPLTPEQLEAVFESLDRAHTGFLTAREFCLGLGMFVGVASAQGANPCRTPEETFESGGLD 12 mcracr2b LPLTPEQLEAVFESLDQAHTGFLTAREFCLGLGKFVGVESAPGGSPLRTSEETFESG ****************:**************** **** ** *..* **.******* hcracr2b VQGTAGSLDEEEEEEERFHTVLEQLGVAPVLGKQRAVRTLWARLQRERPELLGSFEDVLI 18 mcracr2b ---TGGSLEEEEEDVETFYTSLEKLGVARVLGEQWAVRTLWVGLQRERPELLGSLEEVLM 173 *.***:****: * *:* **:**** ***:* ******. ***********:*:**: Coiled-coil hcracr2b RASACLEEAARERDGLEQALRRRESEHEREVRALYEETEQ-LREQSRRPPSQNFARGERR 239 mcracr2b RASACLEAAAREREGLEQALRRRESEHEREVRGLYEELEQQLGEQRHRRQSQNLPREEQR 233 ******* *****:******************.**** ** * ** :* ***:.* *:* hcracr2b SRLELELQSREQDLERAGLRQRELEQQLHAQAAEHLEAQAQNSQLWRAHEALRTQLEGAQ 299 mcracr2b C GHLELELQTREQELERAGLRQRELEQQLQARAAEQLEAQAQHIQLQRAYEAIRAQLDQAQ Q G Q QQ Q Q Q Q Q Q Q Q Q 293.:******:***:***************:*:***:******: ** **:**:*:**: ** hcracr2b EQIRRLESEARGRQEQTQRDVVAVSRNMQKEKVSLLRQLELLRELNTRLRDDRDACEARR 359 mcracr2b EQLSRLEGEAQGRQEQTQRDVVAVSRNMQKEKLSLLRQLELLRELNLRLRDERDACETKL 353 **: ***.**:*********************:************* ****:*****:: hcracr2b AGSSCRKALTTARLPGPTCCCCCC-WARPPRRGSGHLPSAR 399 mcracr2b LGSSHRKALAIAHKPGPIYCCCCCGWARPPRRGSGHLPSAR 394 *** ****: *: *** ***** **************** Figure S5 Exon/intron architecture and cdna sequence of human CRACR2B. (a) Top two schematics represent the exon/intron map of human CRACR2B transcripts. Bottom two schematics show the exon/intron map of the human CRACR2B mrna cloned from Jurkat T cells in comparison with that of mouse CRACR2B (NM_113.2). The mrna sequence of the cdna we cloned is not identical to any predicted transcripts ut showed high similarity with the sequence of mouse CRACR2B. The 5 UTR sequence is not validated and is marked as a dotted line. () The nucleotide sequence of human CRACR2B cdna cloned and sequenced from Jurkat T cells. The start and stop codons are indicated in red. (c) Amino acid sequence alignment of human CRACR2B (cloned from Jurkat T cells) and mouse homologue. Amino acid sequences were aligned using ClustalW2 (default options). The predicted Ca 2+ inding EF-hands in the N terminus are colored in light lue. The residues in red indicate variale regions in the C terminus of CRACR2B that differ significantly with the amino acid sequences of CRACR2A. The predicted coiled-coil region using COILS is oxed in gray.

6 CRAC -R2A GAPDH CRAC -R2B GAPDH Scr R2A Scr R2B Jurkat T cells Scr - + Scr - + R2A: sirna - + :RT R2B - + HEK293 cells Norma alized Expression (rela tive to GAPDH) Scr R2A: sirna Scr R2A: sirna 45 CRACR2A α- CRACR2A (α- myc) 42 α-actin 42 α-actin c HEK293 cells Scr CRACR2A sirna Scr - + Scr CRACR2B sirna CRACR2B - + :shrna RT CRACR2B GAPDH d e HEK293 cells sirna Scr CRACR2A CRACR2B PCR cycles p RT CRACR2A CRACR2B GAPDH 35 Scr Vec shrna: CRACR2B Vec R2A R2B Scr Vec shrna: Vec CRACR2B R2A R2B f [Ca 2+ ] i (nm) Orai1 KO MEFs Ca 2 mm Ca Vector CRACR2A CRACR2B 1µMTG anti-actin Figure S6 Knockdown efficiency of sirnas and shrnas targeting CRACR2A and CRACR2B in Jurkat T cells and HEK293 cells. (a) Jurkat T cells and HEK293 cells were transfected with sirnas targeting CRACR2A or CRACR2B. The mrna levels were visualized y agarose gel electrophoresis (left) and quantified (ar graph) using Multi-Gauge software (Fuji film). GAPDHnormalized mrna levels are plotted relative to those in control cells. In HEK293 cells, CRACR2A transcripts were amplified using higher PCR cycles (4 as compared to 35 for all the other samples). RT stands for addition of reverse transcriptase during cdna synthesis. () HEK293 cells staly expressing Myc-CRACR2A were transfected with scramled sirna (Scr) or sirna targeting CRACR2A. Susequent analysis of CRACR2A expression using immunolot with anti-myc antiody shows decreased CRACR2A expression in sirna treated cells (left). Jurkat T cells transfected with control or CRACR2A-targeting sirna were examined y immunolotting using anti-cracr2a antiody (right). Immunolots with anti-actin antiody were used as a loading control. (c) HEK293 cells staly expressing control (Scr) or CRACR2B-targeting shrna were examined for expression of CRACR2B transcripts. (d) Cross reactivity of sirnas targeting transcripts of CRACR2A and CRACR2B were examined in HEK293 cells. (e) HEK293 cells staly expressing shrna were transfected with plasmids encoding FLAG-tagged CRACR2A or CRACR2B cdnas and immunolotted using anti-flag antiody (left panel). Vec depicts lysates from cells transfected with empty vector. (f) Orai1-null MEFs were transduced with retroviruses encoding empty vector, CRACR2A, or CRACR2B cdnas and SOCE was measured. Each trace is average ± s.e.m of - 35 individual MEFs.

7 Orai1 Orai2 Orai MSLNEHSMQALSWRKLYLSRAKLKASSRTSALLSGFAMVAMVEVQLDADHDY * VTSNHHSVQALSWRKLYLSRAKLKASSRTSALLSGFAMVAMVEVQLETQYQY MGASQHSLRALSWRRLYLSRAKLKASSRTSALLSGFAMVAMVEVQLESDHEY Conserved + residues TM I GST-Orai1 GST-Orai1 Input GST WT R77A K78A R83A K85A K87A SA S89A S9A S93A Input GST WT K85A/ K87A * * WB: anti-cracr2a A WB: anti-cracr2a WB: anti-gst A WB: anti-gst A c tor Vect WT Orai1 K85A A/ K87A tor Vect WT Orai1 K85A A/ K87A tor Vect WT Orai1 K85A A/ K87A Vecto or Orai1 WT K85A/ K87A Vecto or Orai1 WT K85A/ K87A (Orai1) WB: anti-actin WB: anti-myc (CRACR2A) (Orai1) WB: anti-myc (CRACR2A) Input IP with anti-flag (Orai1) Figure S7 The positively charged residues K85 and K87 in the N terminus of Orai1 are important for interaction with CRACR2A. (a) Alignment of the N- terminal residues of human Orai proteins shows conservation of the positively charged lysine and arginine residues (laeled in Red). Arg91 (R91) is the residue mutated to Trp (R91W) in human SCID patients (green). The first transmemrane segment (TM I) and the pore residue, E16 are indicated in lue and asterisk (*), respectively. () GST pulldown assays with wild-type and mutant Orai1 N terminus fragments. In addition to the positively charged residues, the conserved serine residues were examined for their role in CRACR2A inding (left). Pulldown with the N terminus of Orai1 K85A/K87A doule mutant shows minimal inding to CRACR2A (right). Lower panels show the amount of GST fusion proteins using immunolots. All the GSTfused WT, mutant Orai1 fragments, and full-length CRACR2A proteins were purified from E. coli. (c) HeLa cells staly expressing STIM1 were transfected with plasmids encoding Myc-CRACR2A together with either WT Orai1 or Orai1 K85/K87A mutant. After 48 h, cells were treated with 1 µm thapsigargin, cross-linked with DSP and examined y immunolotting for expression of WT Orai1, Orai1 K85/K87A and CRACR2A (Input). Middle panel shows input levels using immunolot with anti-actin antiody. After immunoprecipitation with anti-flag resin, precipitates were analyzed y immunolotting with anti-flag and anti-myc antiodies for detection of Orai1 and CRACR2A, respectively (IP).

8 Vector WT-ec-FLAG K85A/K87A WT-C-FLAG GFP + cells: Surface Staining Permea -ilized Relative Cell Num mer Empty (vector) Orai1-WT-ec-FLAG Cy Empty Orai1-K85A/K87A-ec-FLAG Figure S8 Examination of the expression levels of cells were transfected with a plasmid encoding C- WT Orai1 and Orai1 K85A/K87A proteins. (a) HEK293 terminally FLAG-tagged Orai1 (WT-C-FLAG). Bar, cells expressing either wild-type (WT-ec-FLAG) or 1 μm. () HEK293 cells expressing either WT or Orai1 K85A/K87A mutant (K85A/K87A) with a FLAG Orai1 K85A/87A mutant were permeailized, stained for epitope inserted etween TM III and IV were left Orai1, and examined using flow cytometry. Since unpermeailised (top panel) or permeailised this plasmid also expressed GFP from IRES site, (ottom panel) and immunostained for detection of only GFP + population was gated for analysis. At Orai1. As a control for surface staining, HEK293 least 3, events were collected for each sample.

9 Live cell confocal images: STIM1-YFP Middle Bottom -TG Middle Bottom +TG STIM1-mcherry Middle CRACR2A EF2MUT -GFP Merge Live cell confocal images -TG Bottom -TG Figure S9 Co-expression of CRACR2A EF2MUT enhances clustering of STIM1. (a) Live cell confocal microscopy of a HEK293 cell expressing STIM1- YFP shows localization of STIM1 efore (-TG) and after store depletion (+TG) using 1 µm thapsigargin. Left panels show images of cell from the middle while right panels shows images from the footprint t of the cell to visualize STIM1 clusters. Bar, 5μm. () Live cell confocal microscopy of a HEK293 cell expressing STIM1-mCherry with CRACR2A EF2MUT - GFP shows partial co-localization of STIM1 and CRACR2A from the middle and ottom of the cell without store depletion. Bar, 5 μm. Scale ar in the magnified inset represents 2 μm.

10 Non-reducing Vector CRAC R2A Reducing Vector CRAC R2A Non-reducing Vector CRAC R2A Reducing Vector CRAC R2A TG * * * HW * * * M HW M (STIM1) (STIM1) WB: anti-myc (CRACR2A) WB: anti-myc (CRACR2A) Vector CRACR2A TG c.1 mm Ca 2+ 1 mm Ca P<.5 [Ca 2+ ] i (nm) * * STIM1 Figure S1 Co-expression of CRACR2A induces formation of high MW protein complex of STIM1. HEK293 cells expressing FLAG-STIM1 and Myc- CRACR2A were left untreated or treated with thapsigargin and cross-linked for 1 h using.1 mm DSP. After lysis, cellular extracts were cleared y ultracentrifugation, separated y SDS-PAGE under non-reducing or reducing conditions and immunolotted using anti-flag (STIM1, a) or anti- Myc (CRACR2A, ) antiodies. Magnified inset shows distinct high MW protein ands (HW, indicated as *) of STIM1 after store depletion and presence of similar ands in the samples from cells overexpressing CRACR2A without store depletion. Immunolots under reducing conditions serve as loading controls for STIM1 and CRACR2A. M indicates the monomeric form. (c) Measurement of cytoplasmic [Ca 2+ ] in the asence of ER Ca 2+ depletion. HEK293 cells expressing STIM1 along with various CRACR2A mutants were examined for cytoplasmic Ca 2+ in the presence of Ringer solution containing.1 or 1 mm CaCl 2 ions. * indicate samples with statistically significant differences in.1 and 1 mm Ca 2+ containing solution (P <.5 y t-test). Data represent average ± s.e.m from three independent experiments.

11 2 Ca + 1 nm TG 2 Ca + 1 nm TG Vector [Ca 2+ ] i (nm) [Ca 2+ ] i (nm) CRACR2A EF2MUT [Ca 2+ ] i (nm) Ca + 1 nm TG [Ca 2+ ]i (nm) 2 Ca + 1 nm TG CRACR2A EF2MUT [C Ca 2+ ] i (nm) 2 Ca + 1 nm TG 2 Ca + 1 nm TG 2 µm 2 µm 16 2-APB 16 2-APB [C Ca 2+ ] i (nm) Figure S11 Expression of CRACR2A EF2MUT in Jurkat T cells disrupts Ca 2+ oscillations induced y CRAC channels. (a) Jurkat T cells expressing mcherry (Vector) or mcherry-fused CRACR2A EF2MUT were treated with 1 nm thapsigargin in the presence of 2 mm external Ca 2+ solution to induce asynchronous [Ca 2+ ] i oscillations. Graphs show raw traces of representative individual cells expressing either the empty vector (top, n = 6) or CRACR2A EF2MUT - mcherry (ottom, n =7). () Addition of µm 2- APB inhiits oscillations induced y 1 nm thapsigargin in Jurkat cells expressing CRACR2A EF2MUT. Raw traces of 6 representative individual cells expressing CRACR2A EF2MUT are shown.

12 Fig WB: anti-stim1 WB: anti-stim1 Fig. 2a, left (Orai1) 2 1 (Orai1) WB: anti-cracr2a Re-proe with: anti-stim1 Fig. 2a, right WB: anti-stim1 WB: anti-myc (Orai1) WB: anti-myc (Orai1) (CRACR2A) (CRACR2A) Fig. 2, left WB: anti-cracr2a WB: anti-cracr2a WB: anti-gst Fig. 2, right 2 2 WB: anti-cracr2a WB: anti-gst Figure S12 Full scans of key western lot data

13 Fig. 2c N.S N.S. 2 1 WB: anti-myc WB: anti-myc WB: anti-myc WB: anti-myc 2 (CRACR2A) (CRACR2A) (Orai2) (CRACR2A) (CRACR2A) (Orai3) Fig. 2d 1 min exposure 5 min exposure WB: anti-stim1 (Orai1) (Orai1) WB: anti-cracr2a WB: anti-cracr2a Suppl. Fig WB: anti-gst WB: anti-cracr2a WB: anti-cracr2a WB: anti-cracr2a WB: anti-gst Figure S12 Continued

14 Supplemental Tale 1. Clone name Forward Primer Sequence Reverse Primer Sequence PCR template pegfp-n1-cracr2a CTG CAG AAT TCG GAC CGC CTC GAG ATG GCT CRACR2A in pcdna3.1 myc TGG TCC TTC CGA CCC GCC CCT GAC GGG AGG his vector CAG pegfp-c1-cracr2a AGA TCT CGA GCT ATG GCA GAA TTC TCA GAC CRACR2A in pcdna3.1 myc GCT GCC CCT GAC GGG TGG TCC TTC CGA CCC his vector AGG Cut the CRACR2A fragment pmcherry-n1-cracr2a from pegfp-n1-cracr2a and ligate to pmcherry-n1. peyfp-n1-stim1 CTG CAG AAT TCG CTT CGC CTC GAG ATG GAG CTT AAG AGG CTT CTT STIM1 in pcdna3.1 vector GTA TGC GTC CGT CTT AAA Cut the STIM1 fragment from pmcherry-n1-stim1 peyfp-n1-stim1 and ligate to pmcherry-n1. GCC CTC GAG ATG AGC GTC AGC GGC CGC GAC CRACR2A ΔN (deletion of first 118 CRACR2A in pcdna3.1 myc CAG AAT AAC CCA AGT TGG TCC TTC CGA CCC amino acids) his vector CAG CAG GTC AGC GGC CGC TTC CRACR2A ΔC (deletion of last 128 CGC CTC GAG ATG GCT CRACR2A in pcdna3.1 myc GCC CAT GTC GCC CAG amino acids) GCC CCT GAC GGG AGG his vector ATC CRACR2A 63DAE>AAA TTC TTT CAG ACC TGT GAT GAA GCC CTT GCC CRACR2A in pcdna3.1 myc (CRACR2A EF1Mut GCT GCT GCA GGC AAG TGC AGC AGC ACA GGT ) his vector GGC TTC ATC CTG AAA GAA GTG TTT GAT GCC CTG CAG ATA GCC ATT GCC Site directed mutagenesis CRACR2A 97DAD>AAA GCT GCT GCT GGC AAT AGC AGC AGC CAG GGC (SDM) usingwt CRACR2A GGC TAT CTG ATC AAA CAC cdna as PCR template CRACR2A E18Q TAT CTG ACC CCA CAG AAA TCCAGT AGT GAA SDM using WT CRACR2A CAG TTC ACT ACTGGA CTG CTG TGG GGT CAG cdna as PCR template TTT ATA CRACR2A 97DAD>AAA,E18Q (CRACR2A EF2MUT ) pgex 4T-1 CRACR2A pgex4t-1 WT Orai1 N-terminal fragment (amino acids 64-98) PCR of human CRACR2A PCR of human CRACR2B PCR of human GAPDH PCR of mouse CRACR2A PCR of mouse CRACR2B PCR of mouse GAPDH TAT CTG ACC CCA CAG AAA TCCAGT AGT GAA SDM using CAG TTC ACT ACTGGA CTG CTG TGG GGT CAG CRACR2A 97DAD>AAA cdna as TTT ATA PCR template CGC GAA TTC ATG GCT GCG CTC GAG GAC TGG CRACR2A in pcdna3.1 myc GCC CCT GAC GGG AGG TCC TTC CGA CCC CAG his vector CCG GAA TTC ATG AGC CCG CTC GAG GCC GGA pcdna3.1 myc his-horai1 CTC AAC GAG CAC TCC GAG CAG AGC CGA GGT CTT CAG CCA GAA TAA TTT GGG CTC CAA GTC CCC AAG TCA GG TGT CCA TCA CTG ATG AGG CCC AGG AGC CTC CTT GTC ACA AGG AGG A CAG CAG AAA ATC GTG GAA GGA CTC AGA GGC AGG GAT GAT ATG ACC ACA GTT CTG GA AGA ACG AAC TGG AAT TCC TCT GCT TCT GAT GTG CCC TCA TGA GTC GCT GCA GGA GCA AAC CCA AAT AGC TAG AGC CTT AAG AGA TGT CCT GTG GCT TGG AGA TTG TTG CCA TAG ACT CCA CGA CAT TCA ACG ACC C ACT CAG CAC CG