Western blot was performed as previous study [15 (link)]. Extraction of total cellular proteins was extracted by RIPA buffer (Beyotime, Shanghai, China) containing protease and inhibitor mixes (Thermo Fisher Scientific, New York, USA) on ice. BCA Protein Assay kit (Thermo Scientific, Waltham, MA, USA) was performed to evaluate protein concentration. Equal amounts of proteins were separated by sodium dodecylsulfonate (SDS) polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) membrane by electroblotting (Millipore, Bedford, MA, USA). Primary antibodies were added and incubated throughout a night at 4° C. Primary antibodies including anti-OTUD3 monoclonal antibody (1:500; HPA028543, Sigma), anti-ACTN4 monoclonal antibody (1:1000, 15145, CST). After being incubated with the second antibody (CST, MA, USA) for 1h at room temperature, the intensity of protein bands was evaluated by Quantity One software (Bio-Rad, Hercules, CA, USA).
Hpa028543
Manufactured by Merck Group
The HPA028543 is a laboratory equipment product from Merck Group. It is designed to perform specific functions in a laboratory setting. The core function of this product is to [CORE FUNCTION DESCRIPTION]. No further details or interpretations about the intended use of this product can be provided.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Electroblotting, by Merck Group (2 mentions)
Protease and inhibitor mixes, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Sc 965, by Santa Cruz Biotechnology (1 mentions)
Anti pten, by Cell Signaling Technology (1 mentions)
Anti pser473 akt, by Cell Signaling Technology (1 mentions)
Sc 374171, by Santa Cruz Biotechnology (1 mentions)
Sc 1616, by Santa Cruz Biotechnology (1 mentions)
Sc 25778, by Santa Cruz Biotechnology (1 mentions)
Anti actin, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Tumor tissue microarrays, by Shanghai Outdo Biotech Co. (1 mentions)
Anti gapdh, by ABclonal (1 mentions)
Anti tubulin monoclonal antibody, by Abcam (1 mentions)
6 protocols using hpa028543
1
Western Blot Analysis of OTUD3 and ACTN4
2
Protein Interaction and Truncation Analyses
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Full-length OTUD3 WT, full-length OTUD3 C76A, full-length GRP78, and full-length PTEN were cloned into the pCMV-Myc, pFlag-CMV-2 or pEGFP-C1 vectors as indicated. GST-tagged OTUD3, GST-tagged OTU + UBA (1–340 aa), UBA (184–340 aa), Tail (341–398 aa) truncations were cloned into the pGEX-4T-2 vector. G1 (1–124 aa), G2 (125–399 aa), G3 (281–499 aa), G4 (500–654 aa) truncations of GRP78 were cloned into the pEGFP-C1 vector. Antibodies used in immunoblotting were: anti-actin (1:1,000, sc-1616, Santa Cruz); GAPDH (1:1,000, sc-25778, Santa Cruz);anti-GRP78 (1:1000; 11587-1-AP, Proteintech); anti-OTUD3 (1:500; HPA028543, Sigma); anti-PTEN (1:1000; #9188, Cell Signaling) and anti-pSer473-AKT (1:1000; #4060, CellSignaling); Anti-Flag (1:1000; sc-965, Santa Cruz); anti-Myc (1:1000; sc-374171, Santa Cruz); anti-HA (1:1000; M180-3, MBL); anti-GFP (1:1000; 66002-1-Ig, Proteintech).Normal IgG (sc-2003, Santa Cruz).
3
Subcellular Localization by Immunofluorescence
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For detection of subcellular localization by immunofluorescence, after fixation with 4% paraformaldehyde and permeabilization in 0.2% Triton X-100 (PBS), cells were incubated with antibodies anti-OTUD3 (1:100; HPA028543, Sigma), anti-Calnexin (1:100, ab219644, abcam), anti-PTEN (1:50; 60300-1-lg, Proteintech), anti-GRP78 (1:100,YM1246, Immunoway) for 8 h at 4 °C, followed by incubation with secondary antibody (Invitrogen) for 1 h at room temperature. The nuclei were stained with DAPI (Sigma), and images were visualized with a Zeiss LSM 510 Meta inverted confocal microscope.
4
Quantitative Immunohistochemistry of Cancer Tissues
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Tumor tissue microarrays, purchased from Shanghai Outdo Biotech Company, contain 73 pairs of hepatocellular carcinoma together with matched adjacent normal hepatocellular tissue, 73 pairs of colon cancer together with matched adjacent normal colon tissue, 30 pairs of cervical carcinoma together with matched adjacent normal cervical tissue, and 90 pairs of lung adenocarcinoma together with matched adjacent normal lung tissue and follow-up (range 0–120 months), respectively. Immunohistochemistry was performed by using the avidin-biotin complex method (Vector Labora-tories), including heat-induced antigen-retrieval procedures. Incubation with antibodies against PTEN (1:150; #9188, Cell Signaling), OTUD3 (1:200; HPA028543, Sigma) was carried out at 4 °C for 12 h. All staining was assessed by a quantitative imaging method; the percentage of immunostaining and the staining intensity were recorded. An H-score was calculated using the following formula: H- SCORE = ∑ (PI × I) = (percentage of cells of weak intensity × 1) + (percentage of cells of moderate intensity × 2) + (percentage of cells of strong intensity × 3). PI indicates the percentage of positive cells vs all cells, and I represents the staining intensity.
5
Investigating OTUD3 and OTUD5 Interactions
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OTUDin3 was purchased from ChemDiv (G856-0029). OTUD3 OTU (aa 52-209) and OTUD5 OTU (aa 168-351) recombinant proteins were purchased from SinoBiological. K48-linked Di-Ubiquitin (P20022) and K63-linked Di-Ubiquitin (P20023) were purchased from Solarbio. Antibodies used in the study were anti-GAPDH (1:1000, AC033, ABclonal), anti-OTUD3 (1:500, HPA028543, Sigma), anti-Cleaved-PARP (1:1000, #9544, Cells Signaling Technology), anti-Cleaved-Caspase-3 (1:500, #9661, Cells Signaling Technology), anti-PARP (1:1000, #9532, Cells Signaling Technology), anti-Caspase-3 (1:1000, #9662, Cells Signaling Technology), anti-Ubiquitin (1:1000, #58395, Cells Signaling Technology), anti-GRP78 (1:1000, 11587-1-AP, Proteintech), anti-HA (1:1000, M180-3, MBL), anti-Myc (1:1000, M192-3, MBL), and anti-Flag (1:1000, F7425, Sigma).
6
Western Blot Analysis of Protein Targets
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Western blot was performed as previous study [12] . Extraction of total cellular proteins was extracted by RIPA buffer (Beyotime, Shanghai, China) containing protease and inhibitor mixes (Thermo Fisher Scientific, New York, USA) on ice. BCA Protein Assay kit (Thermo Scientific, Waltham, MA, USA) was performed to evaluate protein concentration. Equal amounts of proteins were separated by sodium dodecylsulfonate (SDS) polyacrylamide gel electrophoresis and transferred onto apolyvinylidene flusoride (PVDF) membrane by electroblotting (Millipore, Bedford, MA, USA). Primary antibodies were added and incubated throughout a night at 4°C. Primary antibodies including anti-OTUD3 monoclonal antibody (1:500; HPA028543, Sigma), anti-ACTN4 monoclonal antibody (1:1000, 15145, CST), anti-p65 monoclonal antibody (1:1000, 8242, CST), anti-p-p65 monoclonal antibody (Ser536)(1:1000, 3033, CST), anti-IκBα monoclonal antibody (1:1000, 4812, CST), anti-p-IκBα monoclonal antibody (Ser32) (1:1000, 2859, CST) and anti-Tubulin monoclonal antibody (code ab7291, 1:2000 dilution, Abcam). After being incubated with the second antibody (CST, MA, USA) for 1h at room temperature, the intensity of protein bands was analyzed by Quantity One software (Bio-Rad, Hercules, CA, USA).
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