Antiphosphorylated plc γ1

Stimulation, extraction, SDS–PAGE, and immunoblotting were performed as previously described (Martin et al, 2014; Izawa et al, 2017). The following antibodies were used for immunoblotting: antiphosphorylated tyrosine (4G10; dilution 1:1,000), antiphosphorylated PLC‐γ1 (#2821S; dilution 1:1,000), anti‐PLC‐γ1 (#2822S; dilution 1:1,000), antiphosphorylated ERK 1/2 (#4376S; dilution 1:1,000), anti‐ERK 1/2 (#4695S; dilution 1:1,000), antiphosphorylated P38 (#4511S; dilution 1:1,000), antiphosphorylated AKT (serine 473, 4058S; dilution 1:1,000), and anti‐Ku70 (4103S; dilution 1:1,000) purchased from Cell Signaling Technology; anti‐CTPS1 (EPR8086B; dilution 1:1,000) purchased from Abcam; and anti‐actin (A2066; dilution 1:1,000) purchased from Thermo Fischer Scientific; anti‐RASGRP1 antibodies from Merck Millipore (MABS146, RASGRP1 epitope recognized unknown; dilution 1:1,000), from Thermo Fischer Scientific (PA5‐25750, raised against 495–521 amino acids of RASGRP1; dilution 1:1,000), and from Abcam (EPR9609, raised against the N‐terminal part of RASGRP1; dilution 1:1,000). Membranes were then washed and incubated with anti‐mouse or anti‐rabbit HRP‐conjugated antibodies from Cell Signaling (dilution 1:10,000). Pierce ECL Western blotting substrate was used for detection.

Total protein was extracted with cell lysis buffer and the protein concentration was quantified using an Enhanced BCA Protein Assay Kit. Protein was separated by 8–10 % SDS-PAGE and electrotransferred to PVDF membranes (Millipore, Billerica, MA, USA). The membrane was blocked for 1 h with 5 % BSA in TBS-T, and probed with corresponding primary antibodies overnight at 4uC, followed by incubation with rabbit and mouse radish peroxidase-coupled secondary antibodies for 1 h. Specific bands were detected using the enhanced chemiluminescence reagent (Millipore, Billerica, MA, USA) on autoradiographic film. The primary antibodies used were as follows: anti-SHH, SHH-neutralization antibody (Abcam, USA), anti-PLCγ1, anti-phosphorylated PLCγ1, anti-ERK1/2, anti-phosphorylated ERK1/2 (Cell Signaling Technology, Danvers, MA, USA), anti-GAPDH (Proteintech, Wuhan, China), PLCγ1 inhibitor (U73122, Sellock, Shanghai, China).