P44 42 mitogen activated protein kinase mapk

Most chemicals, including dexamethasone, 3-isobutyl-1-methylxanthine, insulin, saponin, cycloheximide (CHX), verteporfin, crystal violet, anti-FLAG M2 affinity gels, Dulbecco's modified Eagle medium (DMEM), and antibodies against vinculin (V4505), β-actin (A2228), α-tubulin (T5168), and FLAG-tag (F1804) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Opti-MEM, Lipofectamine 2000, Lipofectamine RNAiMAX, bovine serum albumin, goat serum, 4′,6-diamidino-2-phenylindole (DAPI), and Alexa Fluor-conjugated secondary antibodies were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Antibodies against PIP5Kα (#9693), PIP5Kγ (#3296), phospho-YAP (Ser127; #4911), YAP (#4912), phospho-LATS1 (Ser909; #9157), LATS1(#9153), phospho-TAZ (Ser89; #59971), TAZ (#4883), Merlin (#6995), HA-tag (#3724), Myc-tag (#2278 and #2276), p44/42 mitogen-activated protein kinase (MAPK, #4695), phospho-p44/42 MAPK (Thr202/Tyr204; #8544), p38 MAPK (#9212), phospho-p38 MAPK (Thr180/Tyr182; #9211), c-Jun N-terminal kinase (JNK, #9258), phospho-JNK (Thr183/Tyr185; #4668), Akt (#9272), and phospho-Akt (Ser473; #9271) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against lamin B1 (sc-374015), GAPDH (sc-47724), and green fluorescent protein (GFP, sc-9996) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Equal amounts of protein (30 μg) from each sample were separated on 12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membrane was then blocked with 5% bovine serum albumin in tris-buffered saline containing 0.1% Tween-20 for 1 h at room temperature and incubated overnight at 4°C with anti-succinate dehydrogenase complex, subunit A (SDHA; Abcam, Cambridge, UK), or p44/42 mitogen-activated protein kinase (MAPK; Cell signaling Technology, Beverly, MA, USA) antibodies diluted 1:1000. The membrane was then incubated with horseradish peroxidase-conjugated secondary anti-rabbit antibody (1:1000, Santa Cruz Biotechnology) at room temperature for 1 h. Bands were detected using an enhanced chemiluminescence detection kit (Thermo Scientific, Rockford, IL, USA), after which these blots were re-probed with rabbit monoclonal anti-β-actin antibody (1:1000; Santa Cruz Biotechnology) as a loading control for all experiments. Quantification of immunoreactivity corresponding to the total bands was performed by densitometric analysis using an Image Quant LAS 4000 (Fujifilm, Tokyo, Japan).