Mouse monoclonal anti α tubulin antibody

The following antibodies and dilutions were used for Western blotting: horseradish peroxidase (HRP-linked anti-HA antibody [Roche; 1:5,000]), rabbit polyclonal anti-DHX15 antibody (Bethyl Laboratories, catalog no. A300-390A; 1:15,000), mouse monoclonal anti–α-tubulin antibody (Proteintech; catalog no. 660311-Ig; 1:5,000), HRP anti-mouse (Sigma, catalog no. A9044; 1:5,000), and HRP anti-rabbit (Sigma, catalog no. A9169, 1:10,000). For immunofluorescence, polyclonal rabbit anti-ENP1 (described in ref. 66 (link); kind gift from Ulrike Kutay, ETH Zürich, Zürich, Switzerland; dilution 1:10,000) and monoclonal rat anti-HA (Roche, catalog no. 11867423001; 1:200) were used as primary antibodies, Alexa Fluor 488-labeled goat anti-rabbit (Invitrogen, catalog no. A11008; 1:300) and Alexa Fluor 633-labeled goat anti-rat (Invitrogen, catalog no. A21094; 1:300) were used as secondary antibodies.

Protein extracts were obtained using the lysis buffer (KeyGen Biotech). After being quantified, equivalent amounts of protein extracts were separated using SDS-PAGE and transferred to the PVDF membrane (Roche Applied Sciences). The primary antibody was added onto each membrane and incubated at 4 °C overnight. The appropriate second antibody was used on the following day. Mouse monoclonal anti-SATB2 antibody (1:100, Abcam, United Kingdom), rabbit polyclonal anti-CD133 antibody (1:500, Abnova, China) and mouse monoclonal anti-α-tubulin antibody (1:1000, proteintech, United States) were used. The targeted bands were visualized and photographed with the FluorChem system (Alpha Innotech).