Rabbit anti jmjd3

The protein content of cell extracts or tissue prepared in radioimmunoprecipitation assay lysis buffer was determined using the Bradford protein assay kit (Bio-Rad). About 30 μg of proteins were separated by SDS-polyacrylamide gel electrophoresis. After transferring to nitrocellulose membranes, immunoblots were probed separately with various primary antibodies. Subsequently, the immunoblots were blocked with 5% skimmed milk in Tris-buffered saline solution. Fluorescently labeled or horseradish peroxidase–conjugated secondary antibodies were detected by the Odyssey Infrared Imaging System (LI-COR Biosciences). Primary antibodies were used as following: rabbit anti-JMJD3 (Millipore; catalog no.: 07-1533), rabbit anti-H3K27me3/me2/me1 (Abclonal; catalog nos.: A2363, A2362, and A2361), rabbit anti-UTX (GeneTex; catalog no.: GTX12146), rabbit anti-eNOS (Cell Signaling Technology; catalog no.: 32027), rabbit anti-VE-cadherin (Santa Cruz; catalog no.: sc-28644), mouse anti-α-SMA (Sigma; catalog no.: A5228), goat anti-Hes1 (Santa Cruz; catalog no.: sc-13844), rabbit anti-PCNA (Santa Cruz; catalog no.: sc-7907), mouse anti-β-actin (GeneTex; catalog no.: GTX629630), and mouse anti-GAPDH (Santa Cruz; catalog no.: sc-32233).

Immunostaining was performed on paraformaldehyde (PFA)-fixed paraffin-embedded adult pancreas tissues as previously described [69 (link)]. Primary antibodies included rabbit anti-Mll1 (1/250; Bethyl Laboratories), rabbit anti-Mll3 (1/250; Abcam), rabbit anti-Wdr5 (1/2500; Bethyl Laboratories), rabbit anti-Dpy30 (1/500; Abcam), rabbit anti-Jmjd3 (1/50; Millipore), rabbit anti-Utx (1/2000; Millipore), and guinea pig anti-insulin (1/1000; Abcam). Fluorescently conjugated secondary antibodies included donkey anti-rabbit Alexa 488 (1/1000; Molecular Probes) and goat anti-guinea pig Alexa 546 (1/500; Molecular Probes). Sections were imaged with an SP8 confocal microscope (Leica).