Amersham imagequant 800 imager

Crosslinked samples were mixed with 4× XT sample buffer and 20x XT reducing agent in appropriate volumetric ratios (#1610791 and #1610791 from Bio-Rad). These samples were loaded on 12% Criterion XT Bis–Tris gels (#3450118 Bio-Rad) in XT MOPS buffer (#1610793 Bio-Rad). Gels were run at 20 mA until the samples have completely entered the gel and then at 40 mA until the gel run was complete (typically between 2 and 3 h). Gels were run at room temperature, and additionally in the dark if components contained fluorophores. Gels were scanned for histones H3–H4 and/or PCNA fluorescence (depending on the assay) on an AMERSHAM ImageQuant 800 imager (Cytiva). Bands intensity was calculated using the ROI manager tool in Image J/Fiji and plotted using GraphPad Prism. Where applicable, gels were subsequently stained with Coomassie blue and scanned on AMERSHAM ImageQuant 800 imager (Cytiva).

Small-scale immunoprecipitations were performed in parallel with the main IP to verify Numb IP. Proteins were separated by SDS-PAGE and blotted onto PVDF membranes. The membranes were blocked for 1 hr at 4°C in 50 mM Tris–HCl buffer, 0.15 M NaCl, 1% Tween-20 (TBS) supplemented with 0.5% non-fat dry milk (blocking solution) and incubated overnight at 4°C with a rabbit monoclonal anti-Numb antibody (Cell Signaling #2756; RRID:AB_2154298) diluted 1:1000 in TBS/5% BSA. After washing with TBS, the blot was incubated with HRP-conjugated anti-rabbit IgG (1:8000 dilution in blocking solution, Cytiva NA934; RRID:AB_772206), the blot developed with ECL Prime reagent (RPN2236, Cytiva) and imaged with an Amersham ImageQuant 800 imager (Cytiva).

Cells were collected and centrifuged before the pellet was lysed in a cold RIPPA buffer (Thermo Fisher Scientific, 89901) supplemented with HALT protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, catalog no. 78440). Samples were prepared with NuPAGE LDS Sample Buffer (4X; Thermo Fisher Scientific, catalog no. NP0007) and boiled at 95°C for 5 minutes. Protein samples were quantified using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23225) according to the manufacturer's instructions before equal amounts of proteins were loaded and separated on to 4%–12% NuPAGE or Bolt Bis-Tris protein gels, transferred to nitrocellulose membranes, and blocked with 5% (wt/vol) nonfat dry milk in TBST [20 mmol/L Tris-HCl (pH 7.6), 137 mmol/L NaCl, 0.1% Tween-20]. Membranes were probed with indicated primary antibodies overnight at 4°C. Horseradish peroxidase–conjugated secondary antibodies [Cell Signaling Technology (CST), 7074; 1:2,000] were diluted in 5% (wt/vol) nonfat dry milk in TBST and detected on autoradiographic films or using G:Box gel doc system (Syngene) or Amersham ImageQuant 800 imager (Cytiva) after incubating with the ECL or SuperSignal West Dura reagents (Pierce).