Proteins from cultured cells or mice kidneys were extracted with TRI Reagent (Sigma–Aldrich) according to the manufacturer’s guidelines. Samples were separated by SDS-PAGE and transferred onto PVDF membranes (IPVH00010, Millipore). After blocking the membranes with 5% milk, we used primary antibodies to incubate the membranes overnight at 4 °C against the following proteins: KLF10 (1:1000, ab184182, Abcam), CCNB1 (1:1000, GTX100911, GeneTex), CCND1 (1:1000, GTX108624, GeneTex), PCNA (1:1000, 101239-T46, SinoBiological), KIM1 (1:1000, AF1817, R&D), Anti-FLAG (1:1000, 8146, CST), ACTIN (1:2000, GTX11003, GeneTex), ZBTB7A (1:1000, ab175918, Abcam), PTEN (1:1000, AB170941, Abcam), pAKT (1:1000, 4060 S, CST), AKT (1:1000, 4691 S, CST). The membranes were washed by TBST before incubated with secondary antibodies (1:2000, Jackson ImmunoResearch Inc). The bands of the target proteins were visualized by the LAS-3000 detection system and were quantitively analyzed by Fiji based on ACTIN and control groups respectively.
Gtx100911
Manufactured by GeneTex
Sourced in United States
The GTX100911 is a laboratory equipment designed for scientific research applications. It serves as a core function to assist in the analysis and processing of samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab170941, by Abcam (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Af1817, by R&D Systems (1 mentions)
Ab175918, by Abcam (1 mentions)
Gtx11003, by GeneTex (1 mentions)
Ab3201, by Merck Group (1 mentions)
T6199, by Proteintech (1 mentions)
Mab1864, by GeneTex (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
T7451, by Merck Group (1 mentions)
Bandscan 5.0 system, by Bio-Rad (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (1 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
Pierce ecl western blotting substrate kit, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab124905, by Abcam (1 mentions)
Ab181089, by Abcam (1 mentions)
Bicinchoninic acid protein quantification kit, by Beyotime (1 mentions)
Ab205718, by Abcam (1 mentions)
3 protocols using gtx100911
1
Protein Expression Analysis in Kidney Cells
2
Immunocytochemistry of Cellular Microtubules
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Cells cultured in borosilicate glass Lab‐Tek eight‐well chambers (Nunc) were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) at room temperature for 15 min. Fixed cells were permeabilized with 0.1% Triton X‐100 and then incubated in blocking solution (phosphate‐buffered saline with 2% bovine serum albumin) for 30 min at room temperature. To label cytosolic microtubules, primary ciliary membrane, and axonemal microtubules, cells were incubated for 1 h at room temperature with mouse antibody against α‐tubulin (1:500; Sigma Aldrich, T6199), rabbit antibody against Arl13b (1:500; Proteintech, 17711‐1‐AP), mouse antibody against glutamylated tubulin (1:100; Adipogen, AG‐20B‐0020‐C100), mouse antibody against acetylated tubulin (1:500; Sigma Aldrich, T7451), rat antibody against tyrosinated tubulin (1:100; Sigma Aldrich, MAB1864), rabbit antibody against cyclin B1 (1:500; Genetex, GTX100911), and mouse antibody against detyrosinated tubulin (1:100; MERCK, AB3201) each of which was diluted in blocking solution. Cells were then washed with PBS and incubated for 1 h with appropriate secondary antibodies (1:1,000 dilution; Thermo Fisher) at room temperature.
3
Protein Expression Analysis in Esophageal Cancer
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KYSE-150, TE-10 cells and ESCC cells-formed xenograft tumor tissues were lysed with radioimmunoprecipitation assay lysis buffer (Beyotime). Protein concentrations were assessed using the bicinchoninic acid protein quantification kit (Beyotime). Equal amounts of protein (50 μg) from each sample were separated using 10% SDS-PAGE and electrophoretically transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were closed with 5% BSA at 37°C for 60 min and treated with primary antibodies to GAPDH (1:10000, ab181602, Abcam, Cambridge, MA, USA), KDM5B (1:2000, ab181089, Abcam), PIK3C3 (1:2000, ab124905, Abcam), H3K4me3 (1:1000, ab213224, Abcam), Cyclin B1 (1:2000, GTX100911, GeneTex, Inc., Alton Pkwy Irvine, CA, USA), CDC2 (1:1000, GTX108120, GeneTex), pCDC2 (Tyr15, 1:2000, GTX128155, GeneTex), Beclin-1 (1:1000, #3495S, Cell Signaling Technologies, Beverly, MA, USA), LC-3I/II (1:1000, #12741S, CST), and Bcl-2 (1:1000, #4223S, CST) overnight at 4°C. Subsequently, the membrane was incubated with horseradish peroxidase (HRP)-coupled secondary antibody (1:5000, ab205718, Abcam) for 60 min at room temperature. Protein bands were analyzed using Pierce Western Blotting ECL substrate kit (Thermo Fisher Scientific) and BandScan 5.0 system (Bio-Rad, Hercules, CA, USA).
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