680lt goat anti rabbit igg

LT protein turnover was measured by a cycloheximide (CHX) chase assay using quantitative immunoblot analysis. LT plasmids were co-transfected with an eGFP plasmid to normalize transfection efficiency, and all experiments were performed in triplicate. Cells were lysed in IP buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% TritonX-100, 1 mM PMSF, 1 mM benzamidine) and whole cell lysates without pre-clearing were used for direct immunoblotting. Primary antibodies were incubated overnight at 4 °C, followed by 1 h of secondary antibody incubation at room temperature. All signals were detected using quantitative infrared (IR) secondary antibodies (IRDye 800CW goat anti-mouse, 800CW goat anti-rabbit, 680LT goat anti-rabbit IgG, 680LT goat anti-mouse IgG) (LI-COR); signal intensities were analyzed using a laser-scanning imaging system, Odyssey CLX (LI-COR). CM2B4 (Santa Cruz Biotechnology, Dallas, TX, USA), 2T2 (Millipore, Billerica, MA, USA), PAb416 (Santa Cruz Biotechnology), c-Myc (9E10, Santa Cruz Biotechnology), GFP (D5.1, Cell Signaling) HA-Tag (C29F4, Cell Signaling, Danvers, MA, USA), anti-FLAG (M2, Sigma-Aldrich, St. Louis, MO, USA), anti-α-Tubulin (12G10, DSHB), β-Actin (13E5, Cell Signaling) antibodies were used for this study.

Stably transduced BJ-hTERT cells (selected with puromycin for 14 days) were lysed with an IP buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% TritonX-100, 1 mM PMSF, 1 mM benzamidine) and whole cell lysates were used for immunoblot analyses. Membranes were incubated with a primary antibody overnight at 4 °C, and then incubated with a secondary antibody for 2 h (h) at room temperature (RT). A quantitative infrared (IR) imaging system, Odyssey CLX (LI-COR), was used to determine protein expression. The primary antibodies used in this study included phospho-p53 (Ser15) (Cell Signaling, 9284, Danvers, MA, USA), p21 Waf1/Cip1 (Cell Signaling, 2947), cyclin E (Santa Cruz Biotechnology, sc-247, Dallas, TX, USA), phospho-histone H3 (Ser10) (Cell Signaling, 53348), GFP (Santa Cruz Biotechnology, sc-9996), and β-Actin (Cell Signaling, 4970). All signals were detected using quantitative infrared (IR) secondary antibodies (IRDye 800CW goat anti-mouse, 800CW goat anti-rabbit, 680LT goat anti-rabbit IgG, 680LT goat anti- mouse IgG) (LI-COR).

Western blots were performed using antibodies for anti-FOXM1 (sc-502) and anti-HA (sc-543) and anti-β-actin (ab6276) purchased from Abcam. Fluorescent imaging of GFP-FOXM1 was performed using an anti-GFP antibody (ab290) purchased from Abcam. Cell lysates were prepared using RIPA buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1 % NP-40, 1 % sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na₃VO₄) with proteasome inhibitors (Roche). Lysate was agitated for 30 min at 4 °C by end-to-end rotation, supernatant collected following centrifugation (13,000 rpm, 4 °C, 10 min) and protein concentration measured by BCA (Pierce) assay. Samples loaded onto 4–12 % Tris-Glycine mini gels (Invitrogen), purified by SDS-PAGE and transferred to nitrocellulose membrane (Invitrogen). Membranes were incubated in Odyssey blocking buffer (LiCor) for 1 h at room temperature and probed with FOXM1, HA, or GFP antibody 1:1,000 and β-actin 1:5,000 overnight at 4 °C. For detection, the blot was incubated with LiCor IRDye secondary antibodies; 680LT goat anti-rabbit IgG and 800LT goat anti-mouse IgG both at 1:10,000 and visualized using an Odyssey scanner.