Anti mettl14 hpa038002

The antibodies used in this study were: anti-GAPDH (clone 4G5, AbD serotec, Atlanta, GA), anti-FLAG (F-3165, Sigma-Aldrich), anti-FTO (ab124892, Abcam, Cambridge, MA), anti- AlkBH5 (HPA007196, Sigma-Aldrich), anti-METTL3 (15073-1-AP, Proteintech Group, Rosemont, IL), anti-METTL14 (HPA038002, Sigma-Aldrich), anti-YTHDF1 (ab99080; Abcam), anti-YTHDF2 (ABE542, EMD Millipore, Billerica, MA), anti-YTHDF3 (ab103328; Abcam), and anti-HIV-1 Gag (clone #24–2, the NIH AIDS Reagent Program). Cells were harvested and lysed in cell lysis buffer (Cell Signalling, Beverly, MA) supplemented with protease inhibitor cocktails (Sigma-Aldrich). Immunoblotting was performed as described (St Gelais et al., 2015 (link)). Detection of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) expression was used as a loading control.

The antibodies used in this study were: anti-GAPDH (AHP1628, Bio-Rad), anti-FLAG (F1804, Sigma-Aldrich), anti-METTL3 (15073-1-AP, Proteintech Group), anti-METTL14 (HPA038002, Sigma-Aldrich), anti-FTO (ab124892, Abcam), anti-ALKBH5 (HPA007196, Sigma-Aldrich), anti-MDA5 (D74E4, Cell Signaling), anti-RIG-I (D14G6, Cell Signaling), anti-HIV-1 Gag (clone #24–2, the NIH AIDS Reagent Program), anti-IRF3 (124399, Abcam), anti-phospho-IRF3 (Ser396) (4D4G) (4947, Cell Signaling), anti-IRF7 (4920S, Cell Signaling), anti-phospho-IRF7 (Ser471/472) (5184, Cell Signaling) and anti-m⁶A polyclonal rabbit antibody (202 003, Synaptic Systems). Cells were harvested and lysed in cell lysis buffer (Cell Signaling) supplemented with a protease inhibitor cocktail (Sigma-Aldrich) and a phosphatase inhibitor cocktail (Cell Signaling). Immunoblotting was performed as described [23 (link)]. Detection of GAPDH expression was used as a loading control.

Cells were lysed with RIPA lysis buffer (Solarbio) on ice for 15 mins and centrifugated at 10000 rpm/min at 4°C for 30 mins. The concentration of protein from cell supernatant was quantified using the BCA regent. The equal amount of protein samples was loaded on SDS-PAGE and then transferred to PVDF membrane (Beyotime), blocked with 5% skimmed milk (Solarbio) at room temperature for 2 h and incubated with primary antibody at 4 °C overnight. Primary antibodies were listed as: anti-METTL14 (HPA038002, SigmaAldrich), anti-NANOG (4893, CST), anti-β-catenin (sc-7963, Santa Curz). Afterwards, the PVDF membrane was washed for 3 times with PBS, incubated with primary antibody at room temperature for 1.5 h and the chemiluminescence was detected by Pierce™ ECL Western Blotting Substrate (32106, Thermo Fisher Scientific).