Ca9-22 cells were lysed and equal amounts of protein were loaded and separated on 8% SDS-PAGE gels, and transferred to Hybond 0.2-μm polyvinylidene fluoride membranes. The membranes were blocked and probed with specific primary antibodies at 4°C overnight, followed by secondary antibody incubation for 1 h at room temperature. Membranes were incubated with anti-TAK1 (ab109526; Abcam, Cambridge, UK), anti-MMP-9 (ab119906; Abcam), anti-β4 integrin (ab29042; Abcam), anti-cytokeratin 19 (ab7755; Abcam), anti-p115-rhoGEF (ab220892; Abcam), anti-laminin 5 (ab14509; Abcam), anti-67 kDa laminin receptor (ab133645; Abcam), anti-type IV collagen (ab6586; Abcam), and anti-β-actin (#12,262; Cell Signaling Technology, MA) antibodies for 2 h. Anti-rabbit and anti-mouse IgGs conjugated with horseradish peroxidase were used as the secondary antibodies. Immunoreactivity was detected by ECL. The ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA) was used for detection and analysis of immunoblots.
Ab133645
Manufactured by Abcam
Sourced in United Kingdom
Ab133645 is a recombinant monoclonal antibody that recognizes the protein tyrosine phosphatase, non-receptor type 4 (PTPN4). The antibody is produced in mammalian cells and is purified by protein A affinity chromatography.
Automatically generated - may contain errors
Lab products found in correlation
Ab14509, by Abcam (1 mentions)
Ab7755, by Abcam (1 mentions)
Ab109526, by Abcam (1 mentions)
Ab119906, by Abcam (1 mentions)
Ab29042, by Abcam (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Ab6586, by Abcam (1 mentions)
Ab46798, by Abcam (1 mentions)
Ab133776, by Abcam (1 mentions)
Ab246916, by Abcam (1 mentions)
Ab134961, by Abcam (1 mentions)
Ab97051, by Abcam (1 mentions)
0.45 μm pvdf membrane, by Merck Group (1 mentions)
Ab193349, by Abcam (1 mentions)
Ab179467, by Abcam (1 mentions)
Sc 7482, by Santa Cruz Biotechnology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ab216462, by Abcam (1 mentions)
Eclipse e800, by Nikon (1 mentions)
4 protocols using ab133645
1
Western Blot Analysis of Cell Signaling
2
Comprehensive Western Blot Analysis of Cellular Proteins
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Cellular proteins were extracted with RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein concentration was determined by the BCA method. A total of 40 μg of protein for each group was separated on 12% SDS-PAGE gels and transferred to 0.45-μm PVDF membranes (EMD Millipore). Membranes were then blocked with 5% BSA and incubated with primary antibodies at 4°C overnight against HPS70 (4876, CST), CD9 (ab92726, CST), CD63 (ab193349, Abcam), RBP4 (ab233138, Abcam), RPSA (ab133645, Abcam), RPS3(9538, CST), RPS20 (ab133776, Abcam), RPS14 (ab246916, Abcam), RPL4 (ab234829, Abcam), RPL13 (ab134961, Abcam), HSPD1 (ab46798, Abcam), HSPA8 (8444, CST), P-gp (13342, CST), p-cofilin-1 (3313, CST), cofilin-1 (5175, CST), PP1 (sc-7482, Santa Cruz), PP2A (9780, CST). Anti-β-actin (ab179467, Abcam) and anti-GAPDH (2118, CST) were used as the internal control. Anti-COX IV (11967, CST) was used as loading control for mitochondrial proteins. The membranes were then incubated at 37°C for 1 h with an HRP-conjugated secondary antibody (ab97051, Abcam). Bands were visualized by chemiluminescence according to the manufacturer’s protocols.
3
Immunofluorescence Staining of Cells and Tissues
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Cells were fixed in 4% paraformaldehyde for 15 min after treatment and washed with PBS buffer twice for 5 min each. They were then blocked with 1% bovine serum albumin (BSA) solution at room temperature for 1 h and washed with PBS buffer twice for 5 min each. The cells were then incubated with each of the primary antibodies (anti-CD36, catalog #NB400-144, Novus, USA; anti-Aifm2, catalog #sc-377120, Santa Cruz, CA) overnight at 4 °C and washed. Next, the cells were incubated with a secondary antibody for 1 h, and the nuclei were subsequently counterstained with DAPI for 5 min. Finally, they were observed under a microscope equipped for epi-illumination with 5 × , 20 × , and 50 × objectives (Nikon Eclipse E800). The procedure for kidney tissue immunofluorescence staining was the same as that used for the IHC staining. The samples were incubated with primary antibodies (anti-NGAL, ab216462, Abcam; anti-Laminin, ab133645, Abcam) overnight at 4 °C and a secondary antibody for 1 h at 22 °C. The images were collected and analyzed with a UV microscope equipped with epi-illumination.
4
Lysosomal Profiling of LN-229 Cells
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LN-229 cells were seeded on the coverslips 24 h before the experiments. Lysotracker® (1 μM) was used according to the manufacturer’s instructions for staining of lysosomes. After fixation with 2% paraformaldehyde (PFA), the cells were immunostained with anti-67LR antibody (Abcam #ab133645) and green fluorescent secondary antibody (Thermo Fisher #A11034), followed by counterstaining with DAPI for the nucleus. Images were acquired with an LSM 510 Meta laser confocal microscope system equipped with a 100×/1.4 oil immersion objective lens.
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