β tubulin ab6046

Cells were lysed in 1% NP‐40, 50 mm Tris/HCl pH 7.5, 150 mm NaCl, 0.5% sodium deoxycholate, 1 mm EDTA, cOmplete EDTA‐free Protease Inhibitor Cocktail (Roche/Sigma‐Aldrich, Sydney Australia), PhosSTOP phosphatase Inhibitor cocktail (Roche/Sigma‐Aldrich) and 3 m urea. 15–30 μg of total protein per sample was electrophoresed on NuPAGE™ 4–12% Bis/Tris gels and transferred onto PVDF membranes using iBlot transfer stacks (Invitrogen, Carlsbad, CA, USA). Antibodies used in western blot analysis were phospho‐Akt Ser473 (cat # 4051), AKT1 (2938), PTEN (9559), phospho‐S6 Ser240/244 (5364), S6 (2317), phospho‐4EBP1 T37/46 (2855), 4EBP1 (9644), beclin (3495), phospho‐ERK Thr202/Tyr204 (4370), ERK1/2 (9107) and phospho‐mTOR Ser2448 (2976) from Cell Signalling Technologies (Danvers, MA), β‐tubulin (ab6046, Abcam) and anti‐β‐actin (A5316, Sigma). Secondary antibodies used were fluorescent‐labelled goat anti‐mouse (IRDye(R) 680 CW, LI‐COR, 1 : 15 000) and goat anti‐rabbit (IRDye(R) 800 CW, LI‐COR, 1 : 15 000). Signal was detected using an Odyssey (R) Infrared Imaging System (LI‐COR Biotechnology, Lincoln, NE, USA), and fluorescent intensities were quantified using odyssey software, version 3.0.29 (LI‐COR Biotechnology).

Anti-pSer40/41MCM2 antibody was raised by GenScript (GenScript USA Inc) using a CAPLT(p)S(p)SPGR peptide and further affinity purified. Cleaved PARP (19F4) and cleaved Caspase 3 (5A1E) antibodies were from Cell Signalling Technology; CDC7 (clone SMP171) and β-tubulin (ab6046) antibodies were from Abcam; β-Actin antibody (Cat No. A00702) was from GenScript; MCM2 antibody (Cat No. MCA1859) was from AbD Serotec; pSer41MCM2 antibody (Cat no. A300-117A-1) was from Bethyl Laboratories Inc; γ-H2AX antibody, recognizing pSer139-H2AX (Cat No. 05-636) was from Millipore; pSer108MCM2 antibody was previously described [9] (link). Secondary antibodies labeled with infrared fluorophores (800CW anti-rabbit Cat No. 926-32211, 800CW anti-mouse Cat no. 926-32210, 680LT anti-rabbit Cat No. 926-68021 and anti-mouse Cat no. 926-68020) were obtained from Li-COR Biosciences Ltd, secondary antibodies labeled with TRITC fluorophore were obtained from Jackson ImmunoResearch. Odyssey Infrared Imaging Systems were from Li-COR Biosciences Ltd.