Journal: Cell communication and signaling : CCS

Article Title: A non-catalytic role of IPMK is required for PLCγ1 activation in T cell receptor signaling by stabilizing the PLCγ1-Sam68 complex.

doi: 10.1186/s12964-024-01907-0

Figure Lengend Snippet: Fig. 5 IPMK stabilizes the interaction between Sam68 and PLCγ1. (A) Overexpression co-IP test and immunoblot analysis for PLCγ1, Sam68, and IPMK in Flag-hIPMK transfected HEK293T cells. The cell lysate was immunoprecipitated with anti-Flag M2 beads. (B) Endogenous co-IP test and immunoblot analysis for PLCγ1, Sam68, Src, and IPMK in IPMK KO MEFs. The cell lysates were immunoprecipitated with anti-PLCγ1 antibodies. (C) Overexpression co-IP test and immunoblot analysis for PLCγ1, Sam68, and IPMK in Flag-mIPMK transfected NIH3T3 cells following PDGF (10 ng/mL) stimulation for 3 min after serum starvation for 12 h. The cell lysate was immunoprecipitated with anti-Sam68 antibodies. (D) Endogenous co-IP test and immunoblot analysis for PLCγ1, Sam68, and IPMK in naïve CD4+ T cells. The cell lysate was immunoprecipitated with anti-Sam68 antibodies. All blots presented are representative of at least three independent experiments. Densitometric quantitation results were normalized to control conditions. The normality of all data was ana lyzed using the Shapiro-Wilk test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001, using the one-sample t-test compared to each control

Article Snippet: Antibodies against the following proteins were obtained from the indicated sources: Flag (F1804), Tubulin (T5109) (Sigma Aldrich); Sam68 (sc-1238), normal rabbit IgG (sc-2027), GAPDH (sc-32233), LAT (sc-53550) (Santa Cruz Biotechnology); HA (MMS-101R, Biolegend); GST (2622), β-actin (4970), phospho-PLCγ1 (Tyr783) (2821), PLCγ1 (5690), phospho-Src family (Tyr416) (6943), Src (2108), phosphoPKCθ (Thr538) (9377), PKCθ (13643) (Cell Signaling Technology).

Techniques: Over Expression, Co-Immunoprecipitation Assay, Western Blot, Transfection, Immunoprecipitation, Quantitation Assay, Control

Journal: Cell communication and signaling : CCS

Article Title: A non-catalytic role of IPMK is required for PLCγ1 activation in T cell receptor signaling by stabilizing the PLCγ1-Sam68 complex.

doi: 10.1186/s12964-024-01907-0

Figure Lengend Snippet: Fig. 6 IPMK non-catalytically promotes PLCγ1 phosphorylation. (A) Overexpression co-IP test and immunoblot analysis for PLCγ1, Sam68, and IPMK in GST-hIPMK-DN transfected NIH3T3 cells. The cell lysate was immunoprecipitated with anti-Sam68 antibodies. (B) Immunoblot analysis of PLCγ1 and its phosphorylation (Y783) in GST-hIPMK-DN transfected NIH3T3 cell lysates. (C) In vitro kinase assay and immunoblot analysis using PLCγ1, Sam68, Src, and IPMK WT/KA recombinant proteins. IPMK and ATP were added following the formation of the PLCγ1–Sam68–Src protein complex. *p < 0.05, one-sample t-test compared to control; ns, non-significant, unpaired two-tailed Student’s t-test between His-hIPMK WT and KA. (D) Schematic depiction of PLCγ1– Sam68–Src complex formation for PLCγ1 phosphorylation with or without IPMK. All blots presented are representative of at least three independent experiments. Densitometric quantitation results were normalized to control conditions. The normality of all data was analyzed using the Shapiro-Wilk test. Data are presented as mean ± SD. ns; *p < 0.05, using the one-sample t-test (A-C) and unpaired two-tailed Student’s t-test (C) compared to each control

Article Snippet: Antibodies against the following proteins were obtained from the indicated sources: Flag (F1804), Tubulin (T5109) (Sigma Aldrich); Sam68 (sc-1238), normal rabbit IgG (sc-2027), GAPDH (sc-32233), LAT (sc-53550) (Santa Cruz Biotechnology); HA (MMS-101R, Biolegend); GST (2622), β-actin (4970), phospho-PLCγ1 (Tyr783) (2821), PLCγ1 (5690), phospho-Src family (Tyr416) (6943), Src (2108), phosphoPKCθ (Thr538) (9377), PKCθ (13643) (Cell Signaling Technology).

Techniques: Phospho-proteomics, Over Expression, Co-Immunoprecipitation Assay, Western Blot, Transfection, Immunoprecipitation, In Vitro, Kinase Assay, Recombinant, Control, Two Tailed Test, Quantitation Assay

Journal: Cell communication and signaling : CCS

Article Title: A non-catalytic role of IPMK is required for PLCγ1 activation in T cell receptor signaling by stabilizing the PLCγ1-Sam68 complex.

doi: 10.1186/s12964-024-01907-0

Figure Lengend Snippet: Fig. 8 Graphical summary demonstrating the stabilization of the PLCγ1–Sam68 protein complex by IPMK in TCR-stimulated CD4+ Th cells

Article Snippet: Antibodies against the following proteins were obtained from the indicated sources: Flag (F1804), Tubulin (T5109) (Sigma Aldrich); Sam68 (sc-1238), normal rabbit IgG (sc-2027), GAPDH (sc-32233), LAT (sc-53550) (Santa Cruz Biotechnology); HA (MMS-101R, Biolegend); GST (2622), β-actin (4970), phospho-PLCγ1 (Tyr783) (2821), PLCγ1 (5690), phospho-Src family (Tyr416) (6943), Src (2108), phosphoPKCθ (Thr538) (9377), PKCθ (13643) (Cell Signaling Technology).

Techniques: