Anti wtap antibody

After harvesting MEF WT and WTAP KO cells, they were washed with PBS. Cells were fixed with of 2% Formaldehyde in PBS for 15 min at RT. Subsequently, the cells were permeabilized with 0.5% saponin buffer in PBS. The first antibody was applied for 90 min at RT in 0.5% Saponin buffer in PBS. Along the generated α-m⁶A antibody clones, the α-m⁶A antibody from Abcam (ab151230) was applied. After washing in PBS, the cells were incubated for 30 min at RT with the second antibody (Alex647-conjugated goat α-rat IgG, BioLegend, poly4054 or rabbit IgG, Invitrogen) in 0.5% Saponin buffer in PBS. For detection of the biotinylated antibodies, APC-conjugated streptavidin was applied. To stain WTAP, cells were fixed and permeabilized by using the Foxp3/Transcription Factor staining buffer set (eBioscience) according to manufacturer's instructions. Anti-WTAP antibody (Proteintech, 60188) and FITC-conjugated goat antibody α-mouse IgG (BD Biosciences) were applied. After staining, cells were acquired on a FACS Canto II (BD Biosciences) device and samples were analyzed with FlowJo software.

The following reagents were used: transfection was performed with Lipofectamine RNAiMAX Reagent (Invitrogen Life Technologies, 13, 778-030) according to the manufacturer’s protocol. The following antibodies were used: anti-β-actin antibody (Proteintech, 66009-1-Ig); anti-m6A antibody (Synaptic Systems, 202,003); anti-METTL3 antibody (Abcam, Ab195352, ZEN-BIOSCIENCE [382,974]); anti-METTL14 antibody (Cell Signaling Technology, 51, 104); anti-WTAP antibody (Proteintech, 10200-1-AP); anti-KIAA1429 antibody (SAB, 29, 774); anti-TIMP2 antibody (Abcam, ab180630); anti-NF-κB p65 antibody (Cell Signaling Technology, 8242); anti-phospho-NF-κB p65 antibody (Cell Signaling Technology, 3033); anti-cleaved caspase3 antibody (Abcam, Ab2302); anti-NOTCH3 antibody (Proteintech, 55114-1-AP); anti-NOTCH4 antibody (Affinity, DF13597); anti-WT-1 antibody (Abcam, ab267377); anti-nephrin antibody (Abcam, ab216341); anti-podocin antibody (Abcam, ab181143); anti-MMP2 antibody (Proteintech, 10373-2-AP); anti-MT1-MMP antibody (Proteintech, 14552-1-AP); anti-IGF2BP2 antibody (Proteintech, 11601-1-AP); anti-col-Ⅳ antibody (Abcam, ab6586); and anti-fibronectin antibody (Abcam, ab6586). STZ was purchased from Sigma Chemical Company (MO, USA). The PAS kits were obtained from Solarbio (Beijing, China). Mouse Albumin ELISA Kit was obtained from Abcam Biotechnology (Cambridge, UK).