Anti vinculin h 300

Antibodies used for Western blot analysis included the following: anti-C1orf55/SDE2 (epitope: a.a. 318–410; Sigma-Aldrich), anti-GFP (JL-8, Clontech), anti-HA (6E2, Cell Signaling), anti-pCHK1 (S317, Cell Signaling), anti-Actin (Cell Signaling), anti-CDT1 (Cell Signaling), anti-Flag (M2, Sigma-Aldrich), anti-Tubulin (Sigma-Aldrich), Cyclin E (H-12, Santa Cruz), anti-PCNA (PC-10, Santa Cruz), anti-Vinculin (H-300, Santa Cruz), anti-GST (B-14, Santa Cruz), Cyclin A (H-432, Santa Cruz), anti-γH2AX (JBW301, Millipore), anti-CDT2 (Bethyl), anti-pRPA (S33, Bethyl), pKAP-1 (S824, Bethyl), anti-ORC2 (BD Pharmingen), and anti-MUS81 (MTA30 2G10/3, Abcam). Mitomycin C, camptothecin, hydroxyurea, cycloheximide, aphidicolin, and Z-Leu-Leu-Leu-al (MG132) were purchased from Sigma-Aldrich. Rucaparib (AG-014699) was purchased from Selleckchem. MLN4924, ubiquitin vinyl sulfone, and ubiquitin aldehyde were purchased from Boston Biochem. Drugs were used at the concentrations indicated in the figure legends.

Primary antibodies used were anti-FLAG (MilliporeSigma; catalog no.: F1804, 1:8000 dilution), anti-GFP (Santa Cruz; catalog no.: SC-9996, 1:1000 dilution), anti-GST (GenScript; catalog no.: A00865, 1:2000 dilution), anti-ORC2 (BD Biosciences; catalog no.: 551178, 1:1000 dilution), anti-PCNA (PC10; Santa Cruz; catalog no.: SC-56, 1:4000 dilution), anti-SDE2 (Sigma Atlas; catalog no.: HPA031255, 1:4000 dilution), anti-TIM (Bethyl; catalog no.: A300-961A, 1:2000 dilution), and antivinculin (H-300; Santa Cruz; catalog no.: SC-5573, 1:1000 dilution). Secondary antibodies used were antimouse immunoglobulin HRP (Cell Signaling Technology; catalog no.: 7076, 1:4000 dilution) and anti-rabbit immunoglobulin (Cell Signaling Technology; catalog no.: 7074, 1:4000 dilution). Chemical and reagent information are provided in Table S2.

Cell lysates having equal protein concentrations were loaded on a Criterion TGX 4–20% precast polyacrylamide gel (Bio-Rad) and transferred onto a PVDF membrane. Primary antibodies: anti-CENP-F (ab5) (Abcam), anti-BUB1 (B3), anti-ZW10 (3363C4a), anti-vinculin (H-300), anti-γ-tubulin (C-20), anti α-tubulin (Santa Cruz Biotechnology) and anti β-actin (Sigma Aldrich). HRP-conjugated secondary antibody (Cell Signaling Technology) was used. ImageJ (Fiji software, https://fiji.sc/) were used to quantify western blot signals.