Proteolysis inhibitor

Clonal populations were expanded in 6-well plates (Thermo Scientific) before lysis in NP-40 buffer with proteolysis inhibitor (Sigma). Lysates were prepared by 30 minutes incubation on ice in NP-40 with protease inhibitors (Sigma) before a final 10 minute spin at 14000 rcf. The supernatant was then added to 2x reducing sample buffer and boiled for 5 minutes.
Western Blots were prepared through SDS-PAGE on 4–12% gradient Bolt gels (Thermo Scientific). Gels were run for 15 minutes at 70 V, and 45 minutes at 125 V. Once run, each gel was transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad) using a Turbo transfer system (Bio-Rad). After transfer, membranes were blocked with 4% BSA in 0.1% Tween-20 Tris buffered saline (TBST) and probed with the relevant primary antibodies. Secondary incubations were performed using fluorescent antibodies (LiCor Instruments) in TBST. Anti-mouse 680 and anti-rabbit 800 fluorescent antibodies were used for detection using an Odyssey Fc (LiCor Instruments). Tubulin and GAPDH probes were used as previously described. Anti-CXCR4 antibodies Fusin C8352 (labelled antibody 1) and knock-out validated ab124824 (labelled antibody 2) were obtained from Thermo and Abcam respectively. Finally, Image Studio was used to quantify western blots.

Lysates were prepared from clonal populations of cells in a proteolysis inhibitor (Sigma) before solution in 2x reducing buffer. Western Blots were resolved by SDS-PAGE on 4–12% Bis/Tris gradient NuPage gels (Thermo Scientific). Proteins were transferred to polyvinylidene difluoride (PVDF; Bio-Rad) membranes using a Bio-Rad semi-dry Turbo transfer system. Membranes were blocked with 4% BSA in 0.1% Tween-20 Tris-buffered saline (TBST; 200 mM Trizma base, 1.37 M NaCl, 0.1% Tween-20) and probed with antibodies to α-tubulin (Sigma), GAPDH (loading control) (Abcam) and after antibody stripping with antibodies to eGFP (Sigma) and α-tubulin (Sigma). For quantification of TubA1B expression, wild type lysates (Hek293T, Hel 92.1.7, A549) were first probed with a TubA1B antibody (Abcam) and a β-actin loading control before stripping and re-probing with antibodies to α-tubulin (Sigma) and GAPDH (loading control). Values were normalized to loading controls, and then normalized to brightness to give relative intensity values. All primary antibodies were diluted in the blocking buffer shown above. Secondary incubations were performed in anti-mouse 680 and anti-rabbit 800 fluorescent antibodies (LiCor Instruments) for fluorescent detection using an Odyssey Fc (LiCor Instruments).