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1 Supplemental material THE JOURNAL OF CELL BIOLOGY Gillespie et al., repressor complex induced by p38- Gillespie et al. Figure S1. Reduced fiber size after regeneration in p38- deficient muscle. (A) IPMyoD of extracts harvested at indicated times from CTX-injured C57BL/6 TA muscle with an antibody reactive to phospho-tyrosine and immunoblotted for p38-. (B) Immunoblots showing total protein levels for p38-. (C F) hematoxylin and eosin stained sections of uninjured (C and D) and CTX-injured (E and F) wild-type (C and E) and p38- / (D and F) TA muscle. (G) Fold change in S1
2 Figure S2. Reduced proliferation and elevated apoptosis in p38- deficient satellite cells. (A) Proliferation analysis of wild-type and p38- / satellite cells. Cells were grown and fixed at indicated times under growth conditions and immunostained for Ki67. The number of Ki67-positive cells is reported as a percentage of the total number of DAPI-positive nuclei. Error bars represent ±SEM for n = 3. (B) TUNEL staining of wild-type and p38- / satellite cells after 24 h of proliferation. TUNEL-positive cells are reported as a percentage of the total number of DAPI-positive nuclei. Error bars represent ±SEM for n = 3. (C) Immunoblot analysis of MyHC protein expression from proliferating and differentiating wild-type and p38- / satellite cell derived myoblasts. Tubulin was used as a loading control. (D) Immunoblot analysis, using the indicated antibodies, of C2C12 extracts expressing p38-, MKK6EE and p38-, or empty vector. Black lines indicate that intervening lanes have been spliced out. (E) Immunoblot analysis of differentiating C2C12 myoblasts expressing empty vector or MKK6EE. (F and G) Immunoblot analysis, using the indicated antibodies, of differentiating C2C12 myoblast (F) and 10T1/2 fibroblast (G) extracts expressing MKK6 and/or p38-, or empty vector. MyoDwt and MyoDmut were expressed in 10T1/2 fibroblasts as indicated. Tubulin was used as a loading control. fiber size from wild-type and p38- / TA muscle 21 d after CTX injury. Numbers are normalized to the contralateral TA muscle. Error bars represent standard ±SEM for n = 12. Asterisk denotes significance (P < 0.05). (F) Distribution of fiber sizes from wild-type and p38- / TA muscle 21 d after CTX injury. Contralateral TA is shown as a control. Sizes were determined computationally using Image J software (National Institutes of Health). ptyr, phosphotyrosine. Bar, 100 µm. S2
3 Figure S3. Mechanistic effects of p38- phosphorylation. (A) IP-kinase assays from 10T1/2 fibroblasts expressing MKK6EE and p38-, or empty vector. Extracts were immunoprecipitated with antibody reactive to the Myc tag on p38-, and incubated with the recombinant substrate GST-MyoD (full length), along with GST (negative) and GST-ATF2 (positive) as controls. (B) Endogenous MyoD ChIP assays on differentiating C2C12 myoblast extracts expressing empty vector or MKK6EE alone. (C) ChIP assays on C2C12 myoblast extracts expressing empty vector or p38- DN (T183A/Y185F) mutants after 24 h of differentiation. (D) C2C12 myoblasts expressing p38-, MKK6EE and p38-, or empty vector, were immunoprecipitated for the E protein HEB and immunoblotted for MyoD after 24 h of differentiation. (E) Immunoblots show total protein levels for MyoD and HEB. Tubulin was used as a loading control. (F and G) Endogenous H3K9-2me (F) and KMT1A (G) ChIP assays on differentiating C2C12 myoblast extracts expressing empty vector or MKK6EE alone. PCR was performed on the Igh enhancer as a control. The white line indicates that intervening lanes have been spliced out. (H) Immunoblots from nuclear extracts of 293T cells expressing wild-type or mutant MyoD in the presence or absence of Myc-KMT1A. DN, dominant-negative mutant; H3K9-2me, dimethylated histone H3 Lys9; MyoDmut, MyoD [S199A/S200A] mutant; MyoDwt, wild-type MyoD. MyoD repressor complex induced by p38- Gillespie et al. S3
4 Figure S4. Model demonstrating the biological role of p38- in regulating entry into the myogenic differentiation program. In wild-type muscle, p38- signaling allows for sufficient myogenic precursor amplification before the initiation of differentiation, thereby providing a pool of myocytes available for fusion. However, after satellite cell activation in p38- deficient muscle, myogenic precursors prematurely up-regulate myogenin expression, which impairs proliferation as a consequence of forced initiation of the differentiation program. As a result, the number of myocytes available for fusion is reduced, which prevents efficient myotube formation. S4
5 Figure S5. p38- activity and expression during differentiation. (A and B) Differentiation time course from C2C12 myoblasts showing p38- kinase activity (IP: phospho-tyrosine; immunoblot: p38- ) (A), and p38- and p38- protein expression (B). Tubulin was included as a loading control. (C and D) Coomassie blue stained gels from kinase assays on the N and C termini of MyoD (C), and a MyoD S199A/S200A mutant showing equal loading and expression of recombinant substrates (D). pcdna3 and MKK6EE + Myc p38- refer to the unphosphorylated control and phosphorylation reactions, respectively. Dotted lines indicate that different sized bands from the original blots were placed together in the same panel for ease of viewing using Photoshop software (Adobe). MyoD repressor complex induced by p38- Gillespie et al. S5
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(Supplementary Methods and Materials) GST pull-down assay GST-fusion proteins Fe65 365-533, and Fe65 538-700 were expressed in BL21 bacterial cells and purified with glutathione-agarose beads (Sigma).
Supplemental Data. Sethi et al. (2014). Plant Cell /tpc
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SUPPLEMENTAL FIGURE LEGENDS Suppl. Fig. 1. Notch and myostatin expression is unaffected in the absence of p65 during postnatal development. A & B. Myostatin and Notch-1 expression levels were determined
At E17.5, the embryos were rinsed in phosphate-buffered saline (PBS) and immersed in
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Supplementary Figure S1. Hoechst. cells/field. Myogenin. % of Myogenin+ cells. 0 h chase CONTROL. BrdU. 72 h chase. CONTROL BrdU CONTROL BrdU
Hoechst Myogenin amyhc 24 h 48 h 72 h b cells/field 50 40 30 20 10 0 24h 48h 72h h after plating (4 days growth+ t) c e CONTROL 0 h chase % of Myogenin+ cells h after plating (4 days growth+ t) CONTROL
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A H. sapiens 204 TKLQTS--ERANKLAAPSVSH VSPRKNPSVVIKPEACS-PQFGKTSFPTKESFSANMS LP 259 B. taurus 201 TKLQSS--ERANKLAVPTVSH VSPRKSPSVVIKPEACS-PQFGKPSFPTKESFSANKS LP 257 M. musculus 204 TKSQSN--GRTNKPAAPSVSH
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DOI: 10.1038/ncb3562 In the format provided by the authors and unedited. Supplementary Figure 1 Glucose deficiency induced FH-ATF2 interaction. In b-m, immunoblotting or immunoprecipitation analyses were
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Figure S.6 protein (fasted / control).2.8.4 p47 p97 Rpt Ufd 3 24 48 hours after food deprivation mrn (fasted / control) C mrn (denervated / control) 2.5 2.5.5 3.5 3 2.5 2.5.5 p97 ** Npl4 Ufd p47 2 3 4
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α-cd3 + α-cd28: Time (min): + + + + + + + + + 0 5 15 30 60 120 180 240 300 360 360 n.s. Supplementary Fig. 1 Kinetics of appearence of the faster migrating form of. Immunoblot of lysates from Jurkat cells
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Post-translational modification
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Supplementary information Supplementary Figures Supplementary Figure 1. A. i. HA-JMY expressing U2OS cells were treated with SAHA (6h). DAPI was used to visualise nuclei. ii. U2OS cells stably expressing
S156AT168AY175A (AAA) were purified as GST-fusion proteins and incubated with GSTfused
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Supplemental Materials Supplemental Figure S1 (a) Phenotype of the wild type and grik1-2 grik2-1 plants after 8 days in darkness.
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Supplemental Materials and Methods Co-immunoprecipitation (Co-IP) assay Cells were lysed with NETN buffer (20 mm Tris-HCl, ph 8.0, 0 mm NaCl, 1 mm EDT, 0.5% Nonidet P-40) containing 50 mm β-glycerophosphate,
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SUPPLEMENTARY INFORMATION Supplementary figures Supplementary Figure 1: Suv39h1, but not Suv39h2, promotes HP1α sumoylation in vivo. In vivo HP1α sumoylation assay. Top: experimental scheme. Middle: we
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Figure S1 is related to Figure 1B, showing more details of outer segment of
Supplemental Information Supplementary Figure legends and Figures Figure S1. Electron microscopic images in Sema4A +/+ and Sema4A / retinas Figure S1 is related to Figure 1B, showing more details of outer
Figure legends for supplement
Figure legends for supplement Supplemental Figure 1 Characterization of purified and recombinant proteins Relevant fractions related the final stage of the purification protocol(bingham et al., 1998; Toba
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Supplementary Information Negative regulation of initial steps in skeletal myogenesis by mtor and other kinases Raphael A. Wilson 1*, Jing Liu 1*, Lin Xu 1, James Annis 2, Sara Helmig 1, Gregory Moore
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DOI:.38/ncb327 a b Sequence coverage (%) 4 3 2 IP: -GFP isoform IP: GFP IP: -GFP IP: GFP Sequence coverage (%) 4 3 2 IP: -GFP IP: GFP 33 52 58 isoform 2 33 49 47 IP: Control IP: Peptide Sequence Start
Supplementary Information
Supplementary Information MED18 interaction with distinct transcription factors regulates plant immunity, flowering time and responses to hormones Supplementary Figure 1. Diagram showing T-DNA insertion
Figure S1. Figure S2. Figure S3 HB Anti-FSP27 (COOH-terminal peptide) Ab. Anti-GST-FSP27(45-127) Ab.
/ 36B4 mrna ratio Figure S1 * 2. 1.6 1.2.8 *.4 control TNFα BRL49653 Figure S2 Su bw AT p iw Anti- (COOH-terminal peptide) Ab Blot : Anti-GST-(45-127) Ab β-actin Figure S3 HB2 HW AT BA T Figure S4 A TAG
* ** ** * IB: p-p90rsk. p90rsk (Ser380) (arbitrary units) (Ser380) p90rsk. IB: p90rsk. Tubulin. IB: Tubulin. Ang II (200 nm) Ang II (200 nm)
I: p-p9rsk I: p9rsk I: C I: p-p9rsk I: p9rsk 5 (ka) 5 5 (min) Ang II ( nm) p-p9rsk (Ser8) p9rsk p-p9rsk (Ser8) p9rsk (h) Mannitol 5 mm -Glucose 5 mm p9rsk (Ser8) (arbitrary units) p-p9rsk (Ser8) (arbitrary
The microtubule-associated tau protein has intrinsic acetyltransferase activity. Todd J. Cohen, Dave Friedmann, Andrew W. Hwang, Ronen Marmorstein and
SUPPLEMENTARY INFORMATION: The microtubule-associated tau protein has intrinsic acetyltransferase activity Todd J. Cohen, Dave Friedmann, Andrew W. Hwang, Ronen Marmorstein and Virginia M.Y. Lee Cohen
SUPPLEMENTAL FIGURE LEGENDS. Figure S1: Homology alignment of DDR2 amino acid sequence. Shown are
SUPPLEMENTAL FIGURE LEGENDS Figure S1: Homology alignment of DDR2 amino acid sequence. Shown are the amino acid sequences of human DDR2, mouse DDR2 and the closest homologs in zebrafish and C. Elegans.
Supplementary Materials for
www.sciencesignaling.org/cgi/content/full/8/404/ra120/dc1 Supplementary Materials for The subcellular localization and activity of cortactin is regulated by acetylation and interaction with Keap1 Akihiro
Supplementary Figure S1. The tetracycline-inducible CRISPR system. A) Hela cells stably
Supplementary Information Supplementary Figure S1. The tetracycline-inducible CRISPR system. A) Hela cells stably expressing shrna sequences against TRF2 were examined by western blotting. shcon, shrna
Supplementary Fig. 1 Identification of Nedd4 as an IRS-2-associated protein in camp-treated FRTL-5 cells.
Supplementary Fig. 1 Supplementary Fig. 1 Identification of Nedd4 as an IRS-2-associated protein in camp-treated FRTL-5 cells. (a) FRTL-5 cells were treated with 1 mm dibutyryl camp for 24 h, and the lysates
SUPPLEMENTARY MATERIALS
SUPPLEMENTARY MATERIALS Supplementary Table S1: List of primers used for ChIP analysis and Oligo-pull-down assay. Genes Sequence mppargc1a FW 5 -GCGAGGTTTCTGCTTAGTCA-3 (-2317) RV 5 -ACAATGACTAAGCAGAAACCTCG-3
Supplementary Information
Supplementary Information Supplementary Figures Supplementary Figure 1. MLK1-4 phosphorylate MEK in the presence of RAF inhibitors. (a) H157 cells were transiently transfected with Flag- or HA-tagged MLK1-4