Anti phospho p38 mapk thr180 tyr182

Cell fixation and blocking were performed according to manufacturer instructions. Cells were incubated with antibodies (anti-p21, Cell Signaling Technology (Danvers, MA); anti-p16, Abcam (Cambridge, MA); anti-phospho-p38 MAPK (Thr180/Tyr182), Cell Signaling Technology; anti-γH2AX, Cell Signaling Technology; anti-GM130, BD Bioscience, Clontech, Palo Alto, CA; anti-TGN46, Sigma, St Louis, MO) and with Alexa Fluor 555-labeled secondary antibody F(ab’) fragment (Life Technologies). After washing the cells with PBS, cells were incubated with 1 μg/mL Hoechst 33342 solution for 30 min. Each well was imaged by using IN Cell Analyzer 1000, analyzed by Developer, and visualized by Spotfire DecisionSite Client 8.2 as described above.

The cells were lysed in RIPA lysis buffer (Millipore) containing protease (Bimake) and phosphatase (Bimake) inhibitors. The samples were resolved by SDS-PAGE and transferred to PVDF membrane after blocking, the membranes were incubated with primary antibodies overnight at 4 °C and then exposed to secondary antibodies. Protein bands were visualized using West Femto Substrate Trial Kit (Thermo Scientific). The primary antibodies included anti-p38 MAPK (Cell Signaling Technology), anti-phospho-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology), anti-STAT3 (Abcam), anti-phospho-STAT3 (Tyr705) (Abcam), anti-LC3B (Sigma), anti-Beclin1 (proteintech), anti-β-Tubulin (Cell Signaling Technology), and anti-GAPDH (proteintech). The secondary antibodies were goat anti-mouse IgG HRP (proteintech) and goat anti-rabbit IgG HRP (proteintech).

Human CXCL5/ENA-78 quantikine enzyme-linked immunosorbent assay (ELISA) kit (DX000), recombinant human CXCL5/ENA-78, and anti-CXCR2/CXCL8RB and anti-CXCL5 were purchased from R&D (Minneapolis, MN, USA). SYBR Premix Ex Taq was from TaKaRa (Shiga, Japan). Matrigel™ Basement Membrane Matrix was from BD Bioscience (Franklin Lakes, NJ, and USA). Lipofectamine™ 2000 Transfection Reagent was from Invitrogen (Grand Island, NY, USA). Anti-p44/42 MAPK(Erk1/2), anti–phospho-p44/42 MAPK(Erk1/2) (Thr202/Tyr204), anti-p38 MAPK, anti–phospho-p38 MAPK (Thr180/Tyr182), anti-SAPK/JNK, anti–phospho-SAPK/JNK (Thr183/Tyr185), and ERK1/2 inhibitor U0126 were from Cell Signaling Technology (Boston, MA, USA). CXCR2 inhibitor SB225002 was obtained from Calbiochem (Darmstadt, Germany).

Samples of total proteins were extracted according to the TCA method, as previously described [20 (link)]. Each protein sample was separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose filter. The following antibodies were used in this study: anti-Pgk1 (ab113687, Abcam, Cambridge, UK), anti-Hog1 (#sc-165978, Santa Cruz Biotechnology, Dallas, TX, USA), anti–phospho-p38 MAPK(Thr180/Tyr182) (#4511, cell signaling technology, Danfoss, MA, USA), HRP-conjugated goat anti-rabbit IgG (D110058, BBI, Shanghai, China), and HRP-conjugated goat anti-mouse IgG (D110087, BBI, Shanghai, China).

Anti-OTUD1 (HPA038504, 1:1000) and Anti-OTUD1 (HPA038503, 1:200) were purchased from Atlas Antibodies. Anti-OTUD1 (29921-1-AP, 1:200), anti-ASK1 (67072-1-Ig, 1:1000), Anti-ERK (11257-1-AP, 1:1000), anti-p38 (114064-1-AP, 1:1000), anti-JNK, (66210-1-Ig, 1:1000), anti-HSP70, (10995-1-AP, 1:200), anti-P62 SQSTM1, (18420-1-AP, 1:200), anti-GAPDH, (60004-1-Ig, 1:3000), and anti-Flag tag, (20543-1-AP, 1:1000) were purchased from Proteintech. Anti-phospho-p44/42 MAPK (Erk1/2) Thr202/Tyr204) (#9101, 1:1000), anti-phospho- p38 MAPK (Thr180/Tyr182) (#9211, 1:1000), anti-SAPK/JNK (Phospho-Thr183/Tyr185) (81E11) (#4668, 1:1000), anti-c-Jun (Phospho-Ser73) (D47G9), (#3270, 1:1000), and anti-c-Jun (#9165, 1:1000) were purchased from Cell Signaling Technology. Anti-ASK1 (380952, 1:1000) was purchased from. Anti-ASK1 (Phospho-Thr838) (orb335764, 1:1000) was purchased from Biorbyt. anti-HA tag (2063, 1:1000) and anti-Myc tag (2097, 1:1000) were purchased from DiaAn Biotech. Goat anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab15007, 1:200), Goat anti-Rabbit IgG H&L (Alexa Fluor 555) (ab150078, 1:200), APC Mouse Anti-Human CD44(G44-26) (559942, 1:50), and FITC Mouse Anti-Human CD133(W6B3C1) (567029, 1:50) were purchased from BD Pharmingen. Goat anti- Rabbit IgG H&L (HRP) (BF03008, 1:5000) and Goat anti-Mouse IgG H&L (HRP) (BF03001, 1:5000) were purchased from Biodragon,

Culture media, fetal bovine serum (FBS) and penicillin/streptomycin (P/S) were obtained from Sigma-Aldrich (St. Louis, MO). Anti-PTK6 antibody was obtained from Santa Cruz Biochemistry (Santa Cruz, CA), anti-p44/42 ERK1/2, anti-phospho-p44/42 ERK1/2 (Thr202/Tyr204), anti-p38 MAPK, anti-phospho-p38 MAPK (Thr180/Tyr182), anti-STAT3, and anti-phospho-STAT3 (Tyr705) antibodies were obtained from Cell Signaling Technology (Danvers, MA), and anti-β-actin antibody was obtained from Sigma-Aldrich. The selective ERK1/2 inhibitor U0126 was obtained from Sigma-Aldrich.