Ab3201

Retinoblastoma cells grown in suspension were plated onto poly-D-lysine coated 12-well slides just prior to fixation [48 (link)]. Retinoblastoma cells and R28 cells were fixed with 4% PFA and permeabilized using 0.02% Triton/TBS as described [48 (link)]. Alternatively, cells were fixed and permeabilized with ice-cold methanol followed by acetone at 4°C. Cells were then blocked with 5% NGS in 0.1% BSA/TBS-Tween and incubated overnight at 4°C with primary antibodies in incubating solution containing 1% NGS in 0.1% BSA/PBS-Tween. The antibodies against the following were used: IMPDH1 (Proteintech Cat#22092-1-AP; an antibody raised against the entire IMPDH1 fusion protein), IMPDH2 (Abcam Cat#ab75790), alpha-tubulin (Sigma-Aldrich Cat#T6199), beta-III tubulin (Millipore Cat#MAB1637 and Sigma-Aldrich Cat#T8660), autoantibody It2006 (a kind gift from Edward K. L. Chan), lamin B1 (Abcam Cat#ab16048), detyrosinated tubulin (Millipore Cat#AB3201) and MAP-2 (Abcam Cat#ab32454). Cells were then washed with 0.1% BSA/PBS-Tween and incubated for one hour at 37°C with Alexa fluor® secondary antibodies. Cells were washed with 0.1% BSA/PBS-Tween and mounted with Vectashield Mounting Medium containing DAPI (Vector Laboratories Cat#H-1200) for nuclear staining.

Whole cell extracts were prepared by the standard freeze-thaw technique using LSDB 500 buffer (500 mM KCl, 25 mM Tris at pH 7.9, 10% glycerol (v/v), 0.05% NP-40 (v/v), 16 mM DTT, and protease inhibitor cocktail). Cell lysates were subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and proteins were transferred onto a nitrocellulose membrane. Membranes were incubated with primary antibodies in 5% dry fat milk and 0.01% Tween-20 overnight at 4 °C. The membrane was then incubated with HRP-conjugated secondary antibody against rabbit or mouse as appropriate (Jackson ImmunoResearch) (1/5000 dilution in PBS) for 1 h at room temperature, and visualised using the ECL detection system (GE Healthcare). Antibodies: CHD3 abcam ab84528, CHD4 abcam ab72418, MITF abcam ab3201, SOX10, Abcam, ab155279.