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Raw images of original unedited western blots shown in the main figures and supporting information.

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posted on 08.03.2021, 18:45 authored by Anand Sripada, Kapil Sirohi, Lidia Michalec, Lei Guo, Jerome T. McKay, Sangya Yadav, Mukesh Verma, James Good, Donald Rollins, Magdalena M. Gorska, Rafeul Alam

Fig 1A. Western blot of spleen CD4+ T cells from Tamoxifen-treated Spry2f/f (Spry2+/+) and ERT2-Cre:Spry2f/f (Spry2−/−) mice for Spry2 and actin Fig 1H. Western blot showing Cleaved Caspase-3 (CC-3) levels in splenic CD4+ T cells from Spry2+/+ and Spry2−/− mice. Fig 2A. Immunoblot of basal Spry2 and GAPDH expression in thymocytes (Thy), splenocytes (Spl), naïve (Tn), and memory T cells from B6 mice. Fig 2C. Immunoblot of ERK activation, Spry2 protein induction, and actin expression in unstimulated (US) and stimulated (CD3 and CD3/28) human CD4+ T cells at indicated time points (24 h and 48 h, respectively); P/I represents phorbol myristate acetate and ionomycin. Fig 2G. Immunoblot of Spry2 and transcription factor (T-bet, GATA3, and RORgT) expression in naïve and differentiated CD4+ T cells from C57BL/6 mice (n = 3). Fig 2H. Immunoblot of T-bet, GATA3, and RORγT in CD4+ T cells from Spry2+/+ and Spry2−/− mice. Fig 2J. Immunoblot of p-STAT5 and p-STAT6 in CD4+ T cells, treated with IL-2 (10 ng/mL) and TSLP (10 ng/mL) at indicated time points (in min). * in the blot indicates the location of p-STAT6/STAT6 bands. Fig 6A. p-Tyr and β-Actin immunoblots of sort-purified, anti-CD3/CD28-stimulated CD4+ T cells from Spry2+/+ and Spry2−/− mice. Fig 6B. LCK, ZAP70, CD3ζ, and ERK immunoblots of sort-purified, anti-CD3/28-stimulated CD4+ T cells from Spry2+/+ and Spry2−/− mice. * indicates the location of p-CD3ζ bands. Fig 6F and 6G. p-ERK1/2 and ERK1/2 immunoblots of CD4+ T cells from Spry2+/+ and Spry2−/− mice stimulated with anti-CD3/CD28 or PMA/Ionomycin for 10 min (F) or recombinant IL-33 (20 ng/mL) for 30 min (G). Fig 7C. Immunoblot analysis of total CSK, actin, and Spry2 levels in Spry2+/+ and Spry2−/− CD4+ T cells. Fig 7D. p-CSK and CSK immunoblots of cytosol and membrane fractions of stimulated CD4+ T cells from Spry2+/+ and Spry2−/− mice. Na+ K+ ATPase and Tubulin serve as loading controls for membrane and cytosolic fractions, respectively. Fig 7E. Immunoprecipitation of LCK and immunoblotting of LCK, CSK, and p-CSK in CD4+ T cells from Spry2+/+ and Spry2−/− mice under unstimulated and anti-CD3/CD28-stimulated (10 min) conditions. Fig 8A. Immunoblot of basal Cav-1, Cbp/PAG-1, GAPDH, and Spry2 levels in CD4+ T cells from Spry2+/+ and Spry2−/− mice. Fig 8B. Immunoblots of Cav-1 from cytosol and membrane fractions of stimulated CD4+ T cells from Spry2+/+ and Spry2−/− mice. Na+ K+ ATPase and Tubulin serve as loading controls for membrane and cytosolic fractions, respectively. Fig 8G. Immunoblots of Cav-1, pan-ubiquitin (pan-Ub), and actin from CD4+ T cells of Spry2+/+ and Spry2−/− mice cultured for 14 h in MG132 (5 μM) or chloroquine (50 μM). Fig 8H. p-ERK1/2 and ERK1/2, p-CSK, and CSK immunoblots of CD4+ T cells from Spry2−/− mice treated with a membrane permeable scrambled peptide (AP-ScrP) or Caveolin Scaffolding Domain (AP-CSD). S3A Fig. An immunoblot showing lentiviral-mediated knockdown of endogenous CSK and actin in CD4+ T cells from B6 mice. S3B Fig. Immunoblot of TCR-driven ERK1/2 phosphorylation in CD4+ T cells from Spry2+/+ and Spry2−/− mice transduced Control shRNA (Con shRNA), CSK shRNA1, or CSK shRNA3. S4B Fig. Immunoprecipitation of LCK from murine CD4+ T cells followed by a kinase assay in the presence of recombinant mouse CSK (r-CSK; Lanes: 4, 5, 6, 7 of the Ponceau stained blot) or recombinant mouse Spry2 (r-Spry2; Lanes: 5, 6, 7); WCL represents 5% total cell lysate; IgH and IgL represent Ig heavy and light chains, respectively. Fig 1C and 1E. Gating strategy for the flow cytograms presented in Fig 1C and 1E. Fig 5A. Gating strategy for the flow cytograms for CD4+ T cells from blood PBMCs and bronchoalveolar lavage (BAL) FCS files for Figs 1C, 1E, 1G, 1I, 1J, 1K and 1L and 2D and 2F and S1B, S1C and S1E, S2F and S3 (representing the gating strategy for the cited flow plots), and FCS files for Fig 5A (gating strategy for human CD4+ Spry2+ T cells) are shown in the flow repository database (ID: FR-FCM-Z3G3).

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