Perk1 2 4370s

Cells were gently washed two times in PBS and then lysed in sample buffer 2X. The extracts were sonicated (50 W, 30 s), incubated on ice for 15 min, and boiled for 5 min. The samples were then subjected to 10% SDS‐PAGE. Immunoblots were probed with the following antibodies: pERK1/2 (4370S;1:1000), ERK1/2 (4695;1:1000), P38 (9212S; 1:1000), pP38 (9216S; 1:1000), JNK (9258;1:1000), and pJNK (9251S;1:1000) antibodies were obtained from Cell Signaling. GAPDH (MAB374;1:1000), Tubulin (05–829;1:1000), RAS, and RAS‐GTP (RAS activation kit) antibodies were obtained from Millipore. FBXO42 (ab81638;1:500) was obtained from Abcam.
Blots were developed with HRP‐conjugated anti‐mouse or anti‐rabbit Abs, using SuperSignal West Pico Chemiluminescent Substrate or SuperSignal West Femto Chemiluminescent Substrate from Thermo Scientific. pERK trametinib‐treated Western blots were developed using SuperSignal West Femto Chemiluminescent. Pictures of the blots were taken using Bio‐Rad ChemiDoc MP System. Quantification was done using Image Lab (Bio‐Rad).

The Plvx-GFP plasmid was purchased from Sunny Biotechnology (Shanghai, China). The Plvx-GFP-ALPL expression plasmid was constructed using the following primers: forward: 5′-CCG CTC GAG GCC ACC ATG ATT TCA CCA TTC TTA GTA CTG GCC -3′, reverse: 5′-CCG GAA TTC GGT GAA CAG GAC GCT CAG GGG-3′. The Plvx-GFP-RhoA expression plasmid was constructed using the following primers: forward: 5′-CCG CTC GAG GCC ACC ATG GCT GCC ATC CGG AAG A-3′, reverse: 5′-CCG GAA TTC ACC CAA GAC AAG GCA CCC AGA T-3′. The RhoA promoter-driven luciferase reporter was constructed into PGL3 Basic vector using primers, forward: 5′-CGG GGT ACC ACT TCC TGT ATC CTG TTG TTT GTG T -3′, reverse: 5′-CCC AAG CTT CAA ATG ACA ATG ACA CAG GAC ATA C -3′. The c-Myc plasmid was from our research group [25 (link)]. The antibody against ALPL (GTTX100817) was purchased from Genetex (Irvine, CA, USA). The antibodies against RhoA (2117S), ERK1/2 (4695S), and p-ERK1/2 (4370S) were purchased from Cell Signaling Technology (Beverly, MA, USA). The antibodies against c-Myc (sc-764), RhoA (sc-418) and p-c-Myc (sc-377552) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibody against GAPDH (GTX100118) was purchased from Genetex. 10X Cell Lysis Buffer (9803) was purchased from Cell Signaling Technology.