Eif4a1

The antibodies used were LDH (Santa Cruz, sc-133123), Histone H3 (Abcam, ab1791) Actinin (Santa Cruz, sc-17829), Streptavidin-HRP (Cell Signaling 3999S), eIF3A (Abcam, ab118357), eIF4A1 (Abcam, ab31217), FXR1 (Abcam, ab129089), PABP (Abcam, ab21060), eIF5A (Abcam, ab32443), eIF2α (Invitrogen, AH00802), eIF4G1 (Abcam, ab2609), P53 (Santa-Cruz, sc-126 X), eIF4A3 (Abcam, ab32485), eIF4E (Cell Signaling, 20675), RPS6 (Abcam, ab58350), eIF3D (Abcam, ab12442), S6K1 (Abcam, ab32529), S6K1pT389 (Abcam, ab2571), RPS6pS235 (Abcam, ab12864), KDM5A (Abcam, ab70892). KDM4A and β-Actin antibodies were described in (23 (link)). MCM7 antibody was described in (8 (link)). KDM4A immunoprecipitations were performed with KDM4A-P006.

Cells were washed with PBS, pelleted by centrifugation, and incubated in lysis buffer (150 mM NaCl, 50 mM Tris, pH 7.5; 2 mM EGTA, 2 mM EDTA, 0.3% Tween 20, 0.2% Triton X-100, 25 mM NaF, 25 mM β-glycerophosphate, 0.5 mM NaVO₄, 1X HALT protease inhibitor cocktail [Thermo Scientific], 1X phosphatase inhibitor cocktail 2 [Sigma Aldrich], and 1X phosphatase inhibitor cocktail 3 [Sigma Aldrich]) on ice for 10 minutes. The resulting lysates were cleared by centrifugation at 12,000 rpm for five minutes at 4 °C. The protein concentration of the cleared lysates was determined by BCA protein assay (Thermo Scientific). Equal amounts of protein were separated on 4–20% SDS-PAGE gels (Bio-Rad), transferred to nitrocellulose membranes, and probed with primary antibodies. Antibodies used: BLM (Cell Signaling), eIF4A1 (Abcam), GAPDH (Thermo Scientific), Rad51C (GeneTex), RPA1 (Cell Signaling), β-tubulin (Cell Signaling), and XRCC1 (Cell Signaling). Proteins were visualized by IRDye secondary antibodies (LI-COR) and imaged on an Odyssey CLx (LI-COR). Bands were quantified using Image Studio software (LI-COR).