P38 pt180 py182

Protein containing samples (equivalent of 5 × 10⁶ cells) were loaded onto a 4–15% precast Criterion polyacrylamide gel (Biorad). The separated proteins were transferred onto PVDF membranes (Millipore), and then blocked for 1 h at room temperature in a 1:1 1XPBS:SEA Block buffer (Thermo Scientific). The PVDF membranes were then incubated with primary antibodies against GRB2 (clone 23, Santa Cruz Biotechnology), LAT pY226 (clone J96-1238.58.93, BD Pharmingen), LAT pY132 (Genetex), SLP-76 pY128 (clone J141-668.36.58, BD Pharmingen), ERK1/ERK2 pY187/pT185 (Invitrogen), p38 pT180/pY182 (Cell Signaling), JNK pT183/pY195 (Cell Signaling), Akt pS473 (clone-14-6, Invitrogen), Src pY416 (Cell Signaling), pY783 PLC-γ1 (Cell signaling), PLC-γ1 (Cell Signaling), lymphocyte-specific protein tyrosine kinase (LCK) (Cell Signaling), pY (4G10, Millipore), Gsk3αβ pS21/9 (Cell Signaling), actin (clone C4, Millipore), or GAPDH (Meridian Life Sciences). Secondary antibodies conjugated to IRDye 800CW or IRDye 680 were diluted in SEA Block and incubated with the PVDF membranes for 30 min at room temperature. The membranes were then visualized using the Licor Odyssey Infrared detector.

The following antibodies were used: rabbit monoclonal to PLK3 (clone D14F12, Cell Signaling Technology, #4896); rabbit polyclonal to PLK3 (Novus Biologicals, Abingdon, UK, NBP23-2530); goat polyclonal to PLK3 (Bio-Rad, Hercules, CA, USA, VPA00063); rabbit polyclonal to PLK3 (Sigma-Aldrich, St. Louis, MO, USA, HPA060318); mouse monoclonal to PLK3 (clone B37-2, BD Pharmingen, San Jose, CA, USA, 556518); rabbit polyclonal to PLK3 (St. Johns Laboratory, London, UK, STJ93099); rabbit monoclonal Phospho-PLK1 (Thr210) Antibody (Cell Signaling Technology, Danvers, MA, USA #9062); γ-tubulin (Sigma-Aldrich); GM130, p38-pT180/pY182, cJun-pSer73, phospho-histone H2AX-Ser139 (referred to as γ-H2AX) and HIF1α (Cell Signaling Technology); PPP6R1, PPP6R2 and PPP6R3 (A300-968A, A300-970A, A300-972A, Bethyl Laboratories, Montgomery, TX, USA); 14-3-3, mouse monoclonal to PPP6C and TFIIH (Santa Cruz Biotechnology, Dallas, TX, USA); and rabbit polyclonal to PPP6C (Abcam, Cambridge, UK). HRP-conjugated secondary antibodies were from Bio-Rad, Alexa Fluor-conjugates from ThermoFisher Scientific (Waltham, MA, USA). Etoposide, leptomycin B and nocodazole were from Sigma-Aldrich, calyculin from Santa Cruz Biotechnology.

Samples were processed in at least four replicates per each genotype and loaded on 4–12% Bis-Tris gels (NuPAGE Novex, Thermo Fisher Scientific) and separated at 135 V for 1.5 h. Proteins were then transferred to a PVDF membrane using a Bio-Rad Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA). Membranes were blocked for 1 h at room temperature with 5% bovine serum albumin (BSA) in 0.05% TBS-Tween (TBST) and then incubated with primary antibody at 1:1000 dilution overnight at 4 degrees Celsius. Primary antibodies used here include TNIK (Thermo Fisher Scientific, catalog #PA1-20639), TNIK (Bethyl Laboratories, catalog #A302-695A), Phospho-TNIK – Thr181 (custom antibody), P38 pT180/pY182 (Cell Signaling catalog #4511), JNK1-3 pT183/pY185 (Cell Signaling catalog # 8328), and ERK2 pT202/pY204 (Cell Signaling catalog #4370). Membranes were washed with 0.05% TBST four times, 10 min each, and then incubated with secondary antibodies for 1 h at room temperature. Membranes were washed with 0.05% TBST four times, 5 min each, and imaged using a 4000MM Pro Image Station (Carestream, Rochester, NY).