Total proteins were extracted from CRC tissue and cell samples by the RIPA reagent obtained from Sigma, USA, and separated using 10% SDS-PAGE. The isolated proteins were transferred into PVDF membranes followed by 5% nonfat milk incubation to minimize the noise. Next, the membranes were immersed in milk containing primary antibodies against SOCS3 (Rabbit, 1:2000, ab16030, Abcam, UK), CD133 (Rabbit, 1:1000, ab216323, Abcam), SOX2 (Mouse, 1:2000, ab171380, Abcam), OCT4 (Rabbit, 1:5000, ab109183, Abcam), AKT (Rabbit, 1:500, ab8805, Abcam), STAT3 (Mouse, 1:5000, ab119352, Abcam), p-AKT (Rabbit, 1:1000, ab38449, Abcam), p-STAT3 (Rabbit, 1:5000, ab76315, Abcam), and GAPDH (Rabbit, 1:3000, ab124905, Abcam), and incubated overnight. After excess primary antibodies were washed off by PBS, the membranes were probed by HRP-conjunct secondary antibodies, and the signals were visualized using the ECL reagent (Bio-Rad).
Ab124905
Manufactured by Abcam
Sourced in United Kingdom
Ab124905 is a polyclonal antibody produced in rabbit and recognizes Phosphorylated STAT3 (Tyr705). The antibody is suitable for use in various applications such as Western Blotting and Immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Ecl reagent, by Bio-Rad (2 mentions)
Ripa reagent, by Merck Group (1 mentions)
Ab16030, by Abcam (1 mentions)
Ab216323, by Abcam (1 mentions)
Ab171380, by Abcam (1 mentions)
Ab109183, by Abcam (1 mentions)
Ab38449, by Abcam (1 mentions)
Ab8805, by Abcam (1 mentions)
Ab76315, by Abcam (1 mentions)
Ab119352, by Abcam (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Ab53212, by Abcam (1 mentions)
Nb100 2220, by Novus Biologicals (1 mentions)
Sc 48341, by Santa Cruz Biotechnology (1 mentions)
Vps34, by Abcam (1 mentions)
Bm0002, by Boster Bio (1 mentions)
Beclin 1, by Santa Cruz Biotechnology (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)
4 protocols using ab124905
1
Protein Expression Analysis in CRC
2
Immunofluorescence Analysis of Autophagy in Aortic Smooth Muscle Cells
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The thoracic aortas were isolated and embedded in optimal cutting temperature compound (Sakura, Japan) for sectioning at an 8-um thickness. Frozen slides were incubated overnight at 4ºC with antibodies against LC3B (NB100-2220; 1:100; Novus Biologicals, CO, USA) and alpha-smooth muscle actin (α-SMA) (BM0002; 1:100; Boster Biological Technology) and then treated with FITC-labeled anti-rabbit IgG (31635; 1:100) and Cy3-labeled anti-mouse IgG (A10521; 1:100) secondary antibodies (Invitrogen, CA, USA). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). MASMCs were infected with the mRFP-GFP-LC3 adenovirus for 48 h. The puncta in thoracic aortas and MASMCs were viewed under a confocal microscope (Zeiss LSM800, Carl Zeiss, Munich, Germany) with z-stacks and 63× objective lens. The number of puncta was analyzed using the ImageJ program. For endogenous colocalization studies, MASMCs were incubated with TMEM16A (ab53212; 1:50), VPS34 (ab124905; 1:50) (Abcam), p62 (sc-48402; 1:100), Beclin-1 (sc-48341; 1:1000) (Santa Cruz Biotechnology), and Bcl-2 (BM4985; 1:100) (Boster Biological Technology) antibodies, and processed for colocalization observation with a confocal microscope.
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).
4
PIK3C3 Protein Expression Analysis
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Total proteins of treated ESCC cells were extracted using RIPA buffer (Sigma, USA) following the instructions of manufacturers. After qualifying the density of proteins, 50 μg protein samples were loaded and separated by 10% (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) SDS-PAGE. Next, the target proteins were transferred into polyvinylidene difluoride (PVDF) membranes at −4°C followed by 2 hours incubation with 5% non-fat milk and 8 hours incubation with primary antibodies that against PIK3C3 (Rabbit, 1: 3000, ab124905, Abcam, UK). After washed with PBS for 3 times, the membranes were incubated with horse-radish peroxidase (HRP)-conjunct secondary antibodies and the signals were visible using electrochemiluminescence (ECL) reagent (Bio-Rad). Formalin fixed, paraffin embedded tissues were cut into 5-μm thick sections, then blocked with 5% goat serum and incubated for 20 minutes at room temperature before washed. PIK3C3 was detected by rabbit monoclonal primary antibody (EPITOMICS) at a dilution of 1: 400 for 30 minutes. The sections were subsequently incubated with biotinylated, goat anti-rabbit immunoglobulin (Vector Laboratories, Burlingame, CA) and developed with Vectastain Elite BCA kit (Vector Laboratories) as chromogen according to the manufacture’s recommendation.
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