Anti msk2

An anti‐FLAG antibody (M2) was purchased from Merck Millipore (St. Charles, MO, USA). Anti‐cyclin A (SC‐751), anti‐cyclin D1 (SC‐753), anti‐CDC2 (SC‐163), anti‐CDK4 (SC‐601), anti‐H‐Ras (SC‐520), and anti‐β‐actin (SC‐130301) antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). An anti‐phospho p65/RelA (S276) antibody was purchased from Rockland Immunochemicals (Limerick, PA, USA). Anti‐MSK1 and anti‐MSK2 antibodies were purchased from Cell Signaling Technology (#9309; Danvers, MA, USA) and BD Biosciences (San Jose, CA, USA), respectively. TNFα and PDGF were purchased from PeproTech (Rocky Hill, NJ, USA). LY294002, U0126, SP600125, and SB203580 were obtained from FUJIFILM Wako Pure Chemical (Osaka, Japan). NOX inhibitor, VAS2870, was purchased from Merck Millipore (Burlington, MA, USA).

Lysates were incubated at 95°C for 10 minutes in Laemmli sample buffer (Bio-Rad Laboratories). Lysates were run on an SDS-PAGE gel with a protein ladder standard (Bio-Rad Laboratories) and transferred to a nitrocellulose membrane (Bio-Rad Laboratories). Blots were incubated in 5% milk in TBST for 1 hour at RT. Blots were incubated in primary antibody (1:1,000 anti-MAPK11 Cell Signaling no. 2339, 1:1,000 anti-MAPK14 Cell Signaling no. 8690, 1:1,000 anti-MAPK12 Cell Signaling no. 2307, 1:1,000 anti-MSK2 Cell Signaling no. 3679, 1:5,000 anti-SARS-CoV-1/2 N (1 g/mL, clone 1C7C7, a kind gift from Thomas Moran), 1:3,000 anti-β-actin Cell Signaling no. 3700, 1:1,000 anti-vinculin Cell Signaling no. 13901, 1:1,000 anti-MX1 abcam no. ab95926, 1:1,000 anti-P(Y701)-STAT1 Cell Signaling no. 9167, 1:1,000 anti-STAT1 Cell Signaling no. 14995) in 1% milk in TBST overnight at 4°C. Blots were washed thrice for 5 minutes in TBST. Blots were incubated in secondary HRP-conjugated (Bio-Rad Laboratories) or infrared-conjugated secondary antibodies (LICOR Biosciences) in 1% milk in TBST. Blots were washed thrice for 5 minutes in TBST. Blots were imaged on a Chemiluminescence digital imager (Bio-Rad Laboratories) using FEMTO ECL reagent (Thermo Fisher Scientific) or an infrared digital imager (LICOR Biosciences).