Anti prmt5

INS-1 cells were plated at a density of 2×10⁶ cells/10-cm tissue culture plate, and cultured for 30 hours with each treatment condition. After collection of cells, ChIP assays were performed according to the manufacturer’s instructions using a QuickChIP kit (Novus Biologicals, Centennial, USA), as previously reported (Feng, et al. 2017b (link); Matkar, et al. 2015 (link)). Briefly, cells were fixed with 1% formaldehyde and then lysed according to the protocol in a ChIP lysis buffer with protease inhibitors, and cellular DNA was sheared with sonication. After clearing, this lysate was incubated with either control IgG or a specific primary antibody (4 μg) at 4°C overnight, and collected with protein A/G agarose beads. The antibodies used here include anti-PRMT5 (Cell Signaling Technology, Danvers, USA, Cat # ab31751), anti-BRG1 (Abcam, Cambridge, USA, Cat # ab110641), anti-H3R8 (Abcam, Cambridge, USA, Cat # ab130740), and anti-IgG (Abcam, Cambridge, USA, Cat # ab46540). The protein-DNA complexes were eluted from the beads and DNA was amplified by qPCR using primer pairs (S-Table 1) specific to the insulin promoter. Reactions were done in duplicate, and results are normalized to input chromatin and are reported as % input +/− SD.

Mouse whole pancreas tissue was fixed in 4% formalin and processed for paraffin embedding. Antigen retrieval was performed by microwave heating in antigen retrieval solution-citrate buffered (Abcam, Cambridge, USA). Detection was done using fluorochrome-conjugated secondary antibodies (BD, Franklin Lakes, USA) according to manufacturer’s instructions as previously reported (Yang et al. 2010 (link)). The following primary antibodies were used: anti-Insulin (1:200) (Cell Signaling Technology, Danvers, USA, Cat # 8138S), anti-Prmt5 (1:200) (Cell Signaling Technology, Danvers, USA, Cat # ab31751), and anti-Ki67 (1:200) (Abcam, USA, Cat #ab15580). Cell nuclear staining was performed using DAPI (Sigma-Aldrich, St Louis, USA, Cat # 10236276001). The sections were visualized using a Nikon Eclipse E800 fluorescent microscope (Tokyo, Japan) with a CCD digital camera. Whole pancreas scanning was implemented using a Leica DMI6000B fluorescent microscope (Wetzlar, Germany). Quantification of insulin-positive areas and staining intensity was performed with Image J software (Wayne Rasband, NIH, USA).