Paradkar et al 2024 Western Blot Images
Description
Original western blot images of Figure 2A, Figure 4D, Figure 5C, Supplementary Figure 1A, 2A, 6A from Paradkar et al 2024. The first lane (left lane) is the SeeBlue Plus2 Pre-stained Protein Standard (ThermoFisher Scientific, Cat#LC5925) or the HiMark Pre-stained Protein Standard (ThermoFisher Scientific, Cat#LC5699) in case of Supplementary Figure 1A. Other experimental details and sample names are available here and in the original publication.
Files
Steps to reproduce
For subcellular fractionation experiments, U2OS cells were seeded in 10cm2 plates. Confluent plates were pelleted and subjected to subcellular fractionation according to the manufacturer’s instructions (Thermo Fisher Scientific; Cat#78840). For all other experiments cell pellets were lysed in RIPA containing protease inhibitor cocktail (Roche; Cat#11697498001) and subjected to sonication for 2 minutes with 10 seconds on and 10 seconds off at 100% amplitude. The samples were then spun at 15,000rpm for 10 minutes at 4°C, the supernatant was isolated, and the protein lysates were quantified with Bradford reagent (Bio-Rad; Cat#5000002). Depending upon the abundance of the protein of interest, 25-40µg of lysate was loaded onto NuPAGE 4 to 12%, Bis-Tris gels (Invitrogen; Cat#NP0321). Proteins were transferred onto Immun-Blot PVDF membranes (Bio-Rad; Cat#1620177) using the Mini Trans-Blot Cell (Bio-Rad; Cat#1703930) at 90V for 1.5 hours. The membranes were then blocked in 5% bovine serum albumin (BSA, Sigma; A9418) in 1X Tris-Buffered Saline, 0.1% Tween 20 (TBST, Apex Bioresearch Products; Cat#18-235B) for 1.5 hours at room temperature and primary antibodies were added overnight at the following concentrations: anti-PARP1 (Cell Signaling Technology, 1:1000; Cat#9542), anti-PAR (Millipore, 1:5000; Cat#MABE1031), anti-SIRT6 (Cell Signaling Technology, 1:1000; Cat#12486), anti-Histone H3 (GeneTex, 1:1000; Cat#GTX122148), anti-GAPDH (Proteintech, 1:1000; Cat#60004), anti-PARG (Millipore, 1:500; Cat#MABS61), anti-cleaved PARP (Asp214) (Cell Signaling Technology, 1:1000, Cat#5625), anti-β-tubulin (Cell Signaling Technology, 1:1000, Cat#2146). The blots were washed in TBST and secondary antibodies towards mouse and rabbit were added at a dilution of 1:5000 in TBST and incubated for 1.5 hours. Protein levels were visualized using Clarity ECL substrate (Bio-Rad; Cat#1705061) and images were acquired on the Chemidoc Imaging System (Bio-Rad; discontinued). Band intensity quantification of the subcellular fractionation blots was performed in ImageJ.