For IPs, the S2 cells transfected with specific plasmids were lysed using 1 ml RIPA buffer (Merck, #20-188) containing complete protease inhibitor (Roche, #1697498001) and phosphatase inhibitor (Roche, #4906845001). The protein extracts were incubated with 30 μL HA antibody-coated magnet beads for 6 h at room temperature. The beads were collected and washed six times with lysis buffer, and then detected by western blot. For western blot, antibodies were used at the following concentrations for western blot: mouse anti-α-tubulin at 1:10,000 (Beyotime, #AF0001), mouse anti-V5 antibody at 1:4000 (Invitrogen, # R960-25), rabbit anti-pS6K at 1:1000 (Cell Signaling Technology, #9209), rabbit anti-HA antibody at 1:1000 (Beyotime, #AF0039), guinea pig anti-Nprl3 (generated at this work) at 1:2000.
Mouse anti α tubulin
Manufactured by Beyotime
Sourced in United States, China
Mouse anti-α-tubulin is a primary antibody that specifically binds to the alpha-tubulin subunit of the microtubule protein. This antibody can be used to detect and visualize microtubules in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti v5 antibody, by Thermo Fisher Scientific (1 mentions)
Phosphatase inhibitor, by Roche (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Rabbit anti ps6k, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Zen software, by Zeiss (1 mentions)
Rabbit anti pericentrin, by Abcam (1 mentions)
Mouse anti cenpa, by GeneTex (1 mentions)
Lsm880 system, by Zeiss (1 mentions)
Anti histone h3, by Abcam (1 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (1 mentions)
Anti cox 2, by Abcam (1 mentions)
Anti sp1, by Abcam (1 mentions)
Anti mmp2, by Cell Signaling Technology (1 mentions)
Anti histone h1, by Abcam (1 mentions)
Anti ubiquitin, by Abcam (1 mentions)
Anti cyclin a, by Cell Signaling Technology (1 mentions)
Anti cdk2, by Abcam (1 mentions)
Anti vegf, by Abcam (1 mentions)
Anti cyclin e, by Cell Signaling Technology (1 mentions)
4 protocols using mouse anti α tubulin
1
Immunoprecipitation and Western Blot Analysis
2
Immunostaining of Cytoskeletal and Mitotic Proteins
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Cells plated on coverslips were fixed with 4% paraformaldehyde for 15 min and permeabilized in 0.5% Triton for 10 min at room temperature. The cells were then blocked with 3% bovine serum albumin (BSA) dissolved in PBS for 30 min and incubated with primary antibodies diluted at 1:200 with 3% BSA overnight. The cells were washed with PBS to remove the primary antibodies, followed by incubation with secondary antibodies diluted at 1:200 with 3% BSA for 1 hour. Immuno‐stained samples were then incubated with 0.1 μg/mL 4', 6‐diamidino‐2‐phenylindole (DAPI, Beyotime, Shanghai, China) diluted with PBS for 10 min. After washing with PBS, all immuno‐stained samples were observed and captured using the LSM880 system (Zeiss, Oberkochen, Baden‐Württemberg, Germany). The ZEN software (Zeiss) was used to process and analyze the images. Primary antibodies used were mouse anti‐α‐tubulin (Beyotime, Cat#AT819), rabbit anti‐pericentrin (Cat#ab4448, Abcam, Cambridge, MA, USA), mouse anti‐CENPA (Cat#GTX13939, Genetex, Irvine, CA, USA), rabbit anti‐AURKB (Cat#GTX132702, Genetex, Irvine, CA, USA), rabbit anti‐MCAK (Cat#12139‐1‐AP, Proteintech, Rosemont, IL, USA). Secondary antibodies used were Alexa Fluor 488, 546 (Invitrogen, Waltham, MA, USA).
3
Western Blot Analysis of Cell Extracts
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Cytoplasmic and Nnuclear extracts were obtained using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, USA) following with the manufacturer's instructions. Total cell lysates were prepared with a detergent lysis buffer. Western blots were carried out as previously reported [36 (link), 37 (link)]. The rabbit anti-ING4 (1:2000, Abcam, USA), anti-MMP-2 (1:1000, Cell Signaling Technology, USA), anti-MMP-9 (1:1000, Cell Signaling Technology, USA), anti-VEGF (1:1000, Abcam, USA), anti-COX-2 (1:5000, Abcam, USA), anti-Sp1 (1:5000, Abcam, USA), anti-histone H3 (1:2000, Abcam, USA), anti-ubiquitin (1:1000, Abcam, USA), anti-cyclinA (1:1000, Cell Signaling Technology, USA), anti-cyclinE (1:1000, Cell Signaling Technology, USA), anti-CDK2 (1:2000, Abcam, USA), anti-p21 (1:1000, Cell Signaling Technology, USA), anti-p27 (1:1000, Cell Signaling Technology, USA), anti-histone H1 (1:2000, Abcam, USA) and anti-p53 (1:1000, Santa Cruz Biotechnology, USA) were used for primary antibody incubation at 4°C overnight. The mouse anti-α-tubulin (1:1000, beyotime institute of biotechnology, China) was used for the protein loading control. Each blot was repeated at least three times. The intensity of the protein bands were analyzed by densitometry after normalization to the corresponding protein controls.
4
Protein Extraction and Western Blot
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The eggs laid by y,w flies were collected and cultured on standard food to different developmental stages, and they were then homogenized in an RIPA buffer containing complete protease inhibitors and phosphatase inhibitors (Roche). For the Western blot, antibodies were used at the following concentrations: mouse anti-α-tubulin at 1:10,000 (Beyotime, #AF0001); mouse anti-FKBP39 at 1:2000 (generated in this work). Quantitative measurements of Western blots were performed using ImageJ software.
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