Anti mouse hrp 170 6516

All primary and secondary antibodies used for immunoblotting were used at a 1:1,000 and 1:10,000 dilution, respectively. Anti-O-linked N-acetylglucosamine antibody [RL2] (ab2739) was purchased from Abcam. Antibodies for OGT (AL-34) and OGA (345) were gracious gifts from the Laboratory of Gerald Hart in the Department of Biological Chemistry at the Johns Hopkins University School of Medicine. Actin (A2066) antibody and anti-chicken IgY HRP (A9046) were purchased from Sigma. Chromatin immunoprecipitation (ChIP) grade mouse (G3A1) mAb IgG1 isotype control (5415) and RNA polymerase II antibody, clone CTD4H8 (05-623) were purchased from Cell Signaling Technologies and Millipore, respectively. Anti-rabbit HRP (170-6515) and anti-mouse HRP (170-6516) were purchased from Bio-Rad.
All reagents were purchased form Sigma unless otherwise noted. Cycloheximide (CHX, C7698, Sigma) was used at 50 μg/ml for HeLa cells and 25 μg/ml for K562 cells (19 (link), 20 (link)). Actinomycin D (AMD, A1410, Sigma) was used at 0.5 μg/ml for HeLa cells and 5 μg/ml for K562 cells (20 (link), 21 (link)).

The primary antibodies listed in Supplementary Table 5 were used for immunoblotting. Peroxidase-conjugated goat, anti-rabbit (#170-6515) and anti-mouse (HRP #170-6516) antibodies (Biorad, Germany) were used as secondary reagents.

Protein lysates (40 µg/lane) were mixed with LDS sample buffer X4 (Invitrogen, B0007, X1)/DTT (100 mM) and boiled at 95°C for 5 min before loading. Samples were run at 100 V, transferred using semi-dry transfer at 10 V for 70 min and resolved on PVDF membranes. Equal loading and transfer efficiency were assessed by total protein (REVERT™ 700, Li-cor) staining prior to blocking. Membranes were blocked in 1× TBST containing 3% BSA for 1-2 h at room temperature, and then incubated overnight at 4°C with primary antibody in blocking solution. Membranes were washed and probed with secondary antibody (1:5000; anti-rabbit-HRP, 1706515; anti-mouse-HRP 1706516, Bio-Rad) in blocking solution. Blots were imaged by chemiluminescence (Clarity Max™ ECL, Bio-Rad) using the Gel Doc™ XR+Gel Documentation System (Bio-Rad). Band signal intensities were determined using Fiji ImageJ software. All densitometry values were individually standardized to corresponding values of total protein stain and expressed as the fold difference from the average of the WT group of each blot. Primary antibodies used were as follows: anti-Desmin (1:1000; Sigma-Aldrich, D8281), anti-β-actin (1:1000; Abcam, 8226) and anti-Dnm2 (1:1000; GeneTex, GTX127330).