Mouse anti alpha tubulin

For antibodies, rabbit anti-DISC1 (Millipore, ABN-425) was purchased from Millipore; rabbit anti-BMAL1 (Abcam, ab93806) from Abcam; mouse anti-alpha-tubulin (Proteintech, 66031-1-lg) from Proteintech; rabbit anti-HA (Bethyl, A190-108A) from Bethyl; rabbit anti-GFP (Invitrogen, A11122) from Invitrogen; rabbit anti-Flag (ThermoFisher, PA1-984B) from ThermoFisher; mouse anti-Flag (Sigma, F1804) from Sigma; mouse anti-c-Myc (Santa Cruz, sc-40) from Santa Cruz; GSK3β p-Y216 (Invitrogen, 44-604G) from Invitrogen; mouse anti-GSK3β (Cell Signaling, 9832S) from Cell Signaling. Antibodies were utilized as the first antibody of western blot or precipitating antibody of immunoprecipitation.

Lysates or elution from the oligo purification (OE) from wild-type or mutant telomerase samples were resolved on a 4-12% Bis-Tris NuPAGE gel (Invitrogen, Cat# NP0321BOX) then transferred onto a nitrocellulose membrane. Membranes were blocked for 1 hour at room temperature with 5% non-fat milk, prepared in phosphate buffer saline (PBS) supplemented with 0.2% Tween-20 (PBST), followed by an overnight incubation at 4 °C with primary antibody (1:2,000 rabbit anti-dyskerin antibody, Santa Cruz Biotechnology, Cat# sc-48794, lot E0214, RRID: AB_2091314 or 1:20,000 mouse anti-alpha tubulin, ProteinTech, Cat# 66031-1-Ig, lot 10004185). The following day membranes were washed 3 times with PBST, incubated with a secondary antibody (1:10,000 goat anti-rabbit Alexa-Fluor 680, Abcam Cat# ab175773, lot GR222353-8, or 1:10,000 goat anti-mouse Alexa-Fluor 680, Abcam Cat# ab175775, lot GR3273649-2) for 1 hour at room temperature and then washed 3 additional times with PBST before imaging on a Li-COR Odyssey imager. Densitometry for quantification was performed in ImageJ.

Proteins were extracted from the cell line using Radio Immunoprecipitation Assay (RIPA) buffer (Beyotime Institute of Biotechnology) lysis buffer. The samples were centrifuged at 13000×g for 10 min. at 4 °C, and the supernatants were collected. Proteins were separated using 12.5% SDS-PAGE. After electrophoresis, proteins were transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were blocked with blocking solution (Beyotime Institute of Biotechnology) for 1 h at room temperature. Next, the membranes were incubated using the following primary antibodies overnight at 4 °C: rabbit anti-PTK6 (1:1000, Abcam), rabbit anti-PD-L1 (1:1000; Abclonal) and mouse anti-Alpha tubulin (1:1000, Proteintech). After three washes, the membranes were incubated with HRP Goat Anti-Rabbit IgG (H+L) (1:10,000, Abclonal) for 1 h at room temperature. The blots were then visualized using enhanced chemiluminescent reagent (ECL) (LABLEAD).