Mouse β actin antibody

Total protein lysate (50 µg) was separated on 10% SDS/PAGE, and proteins were probed using rabbit TRPM4 antibody (1 : 500; generated by Pineda, Ozhathil et al., 2018) and mouse β‐actin antibody (1 : 2000; Cell Signaling Technology, Leiden, The Netherlands, #3700). Antibodies and proteins were detected with the LI‐COR (LI‐COR Biosciences, Lincoln, NE, USA) Odyssey Imaging System. Fluorescence of the secondary antibodies IRDye^® Donkey anti‐Mouse (#925‐68022; LI‐COR) and IRDye^® 800CW Goat anti‐Rabbit (#925‐32211; LI‐COR) was quantified using LI‐COR Image Studio Lite software. TRPM4 expression was normalized to β‐actin. Where applicable, statistical significance was analyzed with one‐way ANOVA in graphpad prism 8.

Proteins from the cell lysates were prepared from cell lines as previously described [16 (link)]. Equal amounts of protein were denatured and separated by SDS-PAGE, transferred onto PVDF membranes, and incubated with polyclonal antibodies to TMEM16A (Abcam, ab53212; 1:100) and mouse β-actin antibody (Cell Signalling; 1:500). The peak intensity of each band was visualized using an Enhanced Chemiluminescence kit (Amersham, Little Chalfont, UK) on X-ray film (Millipore Corporation, Billerica, USA).

HEK293T WT cells (0.8 × 10⁶) were seeded in a six-well dish. After 24 h, pX330 plasmid containing the gRNA for the target protein (2 µg) and pcDNA3-FKBP-EGFP-HOTag3 (Addgene, 200 ng) were co-transfected using Lipofectamine 2000 (Thermo Scientific). Cells were FACS-sorted 48 h after transfection. The top 95% of cells with green fluorescent protein (GFP) signals were sorted as single cells into 96-well plates. Surviving colonies were expanded to 6-well plates and then 10-cm plates. KO was confirmed by genomic PCR and western blot with mouse β-actin antibody (Cell Signaling, 1:1000, as a loading control) and either rabbit anti-ALKBH1 (Abcam #128895, 1:2000), anti-TET2 (Novus #NBP2-32104, 0.2 µg/mL) or anti-NSUN2 (Proteintech, #20853-1-AP 1:2000) antibodies.