Mettl14

Human cervical cancer cell line HeLa (CCL-2) and embryonic kidney cell line HEK293T (CRL-11268) were obtained from American Type Culture Collection (ATCC) and were cultured under standard conditions. Control siRNA (1027281, Qiagen), METTL3 siRNA (SI04317096, Qiagen), METTL14 siRNA (SI04317096, Qiagen) or HNRNPC siRNA (10620318, Invitrogen) were transfected into HEK293T cells at a concentration of 40 nM using lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions. Cells were collected 48 hours after the transfection, shock-frozen in liquid nitrogen, and stored at -80 °C for further studies. Western blot analysis using METTL3- (HPA038002, Sigma), METTL14- (HPA038002, Sigma), hnRNP C- (sc-32308, Santa Cruz), GAPDH- (A00192-40, Genescript) specific antibodies was performed under standard procedures. Blotting membranes were stained by ECL-prime (RPN2232, GE Healthcare) and visualized by a digital imaging system (G: BOX, SYNGENE). All synthetic oligos were synthesized by Q.D.

Cells were counted, washed twice with ice-cold PBS and lysed using 1× SDS buffer (100 µL for 1×10⁶ cells). After sonication, equal volumes of lysates were loaded and separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes. Membranes were blocked with 5% non-fat milk (Bio-Rad), incubated sequentially with primary and secondary antibodies and detected by immunoblotting with the Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) or Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare). Antibodies used for Western blotting were as follows: METTL14 (HPA038002, Sigma-Aldrich), MYB (12319, Cell Signaling Technology), MYC (13987, Cell Signaling Technology), HA (11867423001, Roche, Basel, Switzerland), GAPDH (sc-47724, Santa Cruz Biotechnology, Dallas, TX), β-Actin (3700, Cell Signaling Technology). GAPDH or β-Actin was used as a loading control.

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).