Mapkapk2

Western blotting analysis was conducted as previously described (25 (link)). Cells were lysed with RIPA lysis buffer, and total protein concentrations were quantified using Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated on Mini-PROTEAN TGX gels (Bio-Rad) and transferred onto PVDF (polyvinylidene fluoride) membranes (Millipore, Billerica, MA, USA). After blocking with 5% BSA at room temperature for 30 min, the membranes were incubated with the relevant primary antibody followed by an appropriate secondary antibody. The primary antibody of p38, p-p38, MAPKAPK2, p-MAPKAPK2, HSP27 and p-HSP27 were obtained from Cell Signaling Technology (1:2000, Danvers, MA, USA). CNR1 and CNR2 antibody were purchased from Abcam (1:800, Cambridge, MA, USA). Anti-GAPDH (1:10000, Cell Signaling Technology) was used as the loading control. Western blots were developed using ECL reagent (Pierce ECL Western Blotting Substrate, Thermo Scientific).

To separate the cell cytoplasmic and nuclear fractions, cells were first lyzed with low salt buffer (20 mM Hepes pH 7.9, 10% Glycerol, 1.5 mM MgCl2, 0.05 % NP40, protease inhibitors) on ice for 5 minutes and then centrifuged at 4000rpm for 5 minutes at 4°C. Supernatant was collected as cytoplasmic fractions, the pellet was then lyzed with high salt buffer (20 mM Hepes pH 7.9, 10% Glycerol, 1.5 mM MgCl2, 500mM NaCl, 0.05 % NP40, protease inhibitors) to collect the nuclear fractions. Co-immunoprecipitation and Western blot were performed as previously described (Wang et al., 2014a (link)). The following antibodies were used for Western blotting: METTL3 (Proteintech,15073-1-AP); β-Tubulin (Abcam, ab6046); EGFR (Cell Signaling Technology, #2232); TAZ (BD Pharmingen, 560235); MAPKAPK2 (Cell Signaling Technology, #3042); DNMT3A (Abcam, ab13888); β-Actin (Abcam, ab119716); Fibrillarin (Abcam, ab4566); CBP80 (Choe et al., 2012 (link)); CTIF (Choe et al., 2012 (link)); eIF4E (Cell signaling technology, #2067); eIF3b (Santa Cruz Biotechnology,sc-16377); eIF4AI (Abcam, ab31217); eIF4GI (Cell signaling technology, #2498); FLAG (Sigma, A8592); METTL14 (Sigma, HPA038002); WTAP (Proteintech Group 60188-1-Ig); YTHDF1 (Abcam, ab99080); YTHDF2 (Proteintech, 24744-1-AP).

ELISA for IFN-γ and IL-12p70 production was performed by ELISA kits (R&D Systems). For Westernblot, anti-mouse p38α (Cat#9218), p38γ (Cat#2307), p38 (Cat#8690), p-p38 (Cat#4631), MAPKAPK-2 (Cat#3042), pMAPKAPK-2 (Cat#3041), RelA (Cat#4764), RelB (Cat#4954), p52 (Cat#4882), GAPDH (Cat#2118), PARP (Cat#9532), C/EBP-β (Cat#3087), pC/EBP-β (Cat#3084), PPARγ (Cat#2430), pStat3 (Cat#9145), Stat3 (Cat#12640), pSmad3 (Cat#9520), Smad3 (Cat#9523), IκB-α (Cat#4812) and β-actin (Cat#4970) from Cell Signaling (1:1000 dilution), and anti-mouse p38β, (Cat#sc-6187), p38δ,(Cat#sc-7587), p50 (Cat#sc-7178) and c-Rel (Cat#sc-70) from Santa Cruz Biotechnology (1:250 dilution) were used.