Western blot analyses were performed as previously described [53 (link)]. Antibodies used were anti-RUVBL1 (sc-15259, Santa Cruz, 1:500), anti-RUVBL2 (gift from Matthias Gstaiger, ETH Zurich, 1:1000), anti-βTubulin (mouse monoclonal sc-5274, Santa Cruz, 1:1000), anti-TFIIH (rabbit polyclonal sc-293, Santa Cruz, 1:4000), anti-FLAG (F-3165, Sigma, 1:20000) and anti-GFP (mouse monoclonal, sc-9996, Santa Cruz, 1:500).
Sc 9996
Manufactured by Santa Cruz Biotechnology
Sourced in United States, Germany, United Kingdom
Sc-9996 is a laboratory equipment product offered by Santa Cruz Biotechnology. It is designed for general laboratory use, but the core function and specific intended use of this product are not detailed in the information provided. A concise and unbiased description cannot be presented without risk of extrapolation or interpretation.
Automatically generated - may contain errors
Lab products found in correlation
F3165, by Merck Group (8 mentions)
Sc 47778, by Santa Cruz Biotechnology (6 mentions)
Anti gfp, by Santa Cruz Biotechnology (5 mentions)
Protease inhibitor cocktail, by Merck Group (5 mentions)
Anti gfp antibody, by Santa Cruz Biotechnology (4 mentions)
T5168, by Merck Group (3 mentions)
Sc 7392, by Santa Cruz Biotechnology (3 mentions)
Sc 40, by Santa Cruz Biotechnology (3 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (3 mentions)
Irdye 800cw goat anti mouse, by LI COR (3 mentions)
Sc 138, by Santa Cruz Biotechnology (3 mentions)
Anti flag, by Merck Group (2 mentions)
Sc 216, by Santa Cruz Biotechnology (2 mentions)
Ab32157, by Abcam (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Cpsf6, by Fortis Life Sciences (2 mentions)
Dazap1, by Fortis Life Sciences (2 mentions)
Alexa flour 488 goat anti mouse, by Thermo Fisher Scientific (2 mentions)
Flouromount g, by Southern Biotech (2 mentions)
Dm5500 b upright automated microscope, by Leica (2 mentions)
126 protocols using sc 9996
1
Western Blot Analysis of RUVBL1/2
2
Immunoblot Analysis of GFP and AtEXPA10
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Protein extraction and immunoblot analyses were performed as previously described in Lee et al. (2013 (link)). Immunoblots were probed with an anti-GFP antibody (Sc9996, Santa Cruz Biotechnology, CA; 1:3000 dilution) and an anti-AtEXPA10 antibody (ABIN678788, Bioss Antibodies, MA; 1:1000 dilution). Polypeptide density measurements used for preparing the histogram of α-expansin/cytosolic fructose 1,6 bisphosphatase ratios were made with ImageJ.
3
Cell Lysis and Western Blot Protocol
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Cells were lysed for 30 min at 4 °C with radio-immunoprecipitation assay (RIPA) buffer (50 mM Tris, pH 7.2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 10 mM MgCl2, 5% glycerol) containing 1% protease inhibitor (Sigma-Aldrich; St. Louis, MO, USA). Lysate was centrifuged at 14,000× g for 15 min at 4 °C. The supernatant was collected and the protein concentration was determined with a BCA assay (Beyotime).
For Western blot, proteins (30 μg for total cell proteins; 10 μg for sEV proteins) were separated with SDS-PAGE, and then the gel was transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad; Hercules, CA, USA). The membranes were blocked with 3% (w/v) bovine serum albumin (BSA, Beyotime) in TBST for 30 min at 37 °C, and probed with primary antibodies (1:1000, v/v) against GFP (sc-9996, Santa Cruz Biotechnology, CA, USA); FAK (610087), pFAK (611722) (BD Biosciences, San Jose, CA, USA); Alix (2171s), Calnexin (2433s) (Cell signaling Technology, Danvers, MA, USA); GAPDH (G9545, Merck, Darmstadt, Germany); and integrin β1 (ab30388), TSG101 (ab83), CD63 (ab134045) (Abcam, Cambridge, UK) overnight at 4 °C and incubated with an appropriate horse radish peroxidase (HRP)-conjugated secondary antibody (1:5000, v/v) for 30 min at 37 °C. Bands were visualized using enhanced chemiluminescence (Vazyme Biotech, Nanjing, China).
For Western blot, proteins (30 μg for total cell proteins; 10 μg for sEV proteins) were separated with SDS-PAGE, and then the gel was transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad; Hercules, CA, USA). The membranes were blocked with 3% (w/v) bovine serum albumin (BSA, Beyotime) in TBST for 30 min at 37 °C, and probed with primary antibodies (1:1000, v/v) against GFP (sc-9996, Santa Cruz Biotechnology, CA, USA); FAK (610087), pFAK (611722) (BD Biosciences, San Jose, CA, USA); Alix (2171s), Calnexin (2433s) (Cell signaling Technology, Danvers, MA, USA); GAPDH (G9545, Merck, Darmstadt, Germany); and integrin β1 (ab30388), TSG101 (ab83), CD63 (ab134045) (Abcam, Cambridge, UK) overnight at 4 °C and incubated with an appropriate horse radish peroxidase (HRP)-conjugated secondary antibody (1:5000, v/v) for 30 min at 37 °C. Bands were visualized using enhanced chemiluminescence (Vazyme Biotech, Nanjing, China).
4
Whole-mount Immunofluorescence Assay Protocol
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Whole-mount immunofluorescence assay was performed essentially as described previously (Zhang et al., 2012 (link); Xue et al., 2014 (link)). Primary antibodies for immunofluorescence were: mouse anti-GFP (1:200; sc-9996, Santa Cruz), rabbit anti-GFP (1:800; Ab290, Abcam), mouse anti-acetylated tubulin (1:400; T6793, Sigma), rabbit anti-aPKC (1:100; sc-216, Santa Cruz), rabbit anti-pH3 (1:200; #9701, Cell Signaling Technology), rabbit anti-active capase3 (1:200; BD 559565, BD Biosciences), mouse anti-GST (1:400; BE2075, EASYBIO), rabbit anti-p-MLC2 (Thr18/Ser19) (1:200; #3674, Cell Signaling Technology), and rabbit anti-RhoA (1:500; BS1782, Bioworld) antibodies. Secondary antibodies were Alexa Fluor 488- or 649-conjugated anti-mouse IgG (1:100; 115-545-003 and 115-605-003, Invitrogen) and Alexa Fluor 488- or 649-conjugated anti-rabbit IgG (1:100; A11008 and A27040, Invitrogen) antibodies. For imaging, the embryo region containing DFCs or KV was dissected, embedded in the mounting medium and were observed under a Zeiss LSM META confocal microscope. Confocal images of DFCs were acquired as a z-series with a step-size of 2 μm and those of KV as a z-series with a step-size of 1 μm.
5
Antibody Generation and Reagent Validation
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Rabbit polyclonal antibody against EV-A71 3A was prepared as described (Tang et al., 2007 (link)). Mouse monoclonal antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (G8795) and Flag (F1804) were purchased from Sigma-Aldrich (St Louis, MO, USA). Rabbit monoclonal antibodies against phospho-eIF2α (#3597) and PKR (Ab32052, against amino acids 50–150 of human PKR) were obtained from Cell Signaling (Beverly, MA, USA) and Abcam (Cambridge, UK), respectively. Mouse monoclonal antibody against green fluorescent protein (GFP) (SC-9996) and rabbit polyclonal antibodies against eIF2α (SC-11386) and poly (ADP-ribose) polymerase (PARP) (SC-7150) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal antibody against EV-A71 3C and rabbit polyclonal antibody against phospho-PKR were obtained from GeneTex (Irvine, CA, USA). Poly(I:C) and 2-aminopurine (2-AP) was purchased from Invivogen (San Diego, CA, USA). Staurosporine was purchased from Sigma-Aldrich.
6
Retroviral Expression of GFP and ORFV073-GFP
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A retroviral expression system (pLNCX2; Clontech) was used to construct HeLa cells constitutively expressing GFP (GFP/HeLa) or ORFV073-GFP (ORFV073GFP/HeLa) fusion protein. GFP or ORFV073-GFP DNA sequences were cloned into plasmid pLNCX2 and transfected into the packaging cell line GP2-293 using Lipofectamine 2000. After 48 h, supernatants containing GFP or ORFV073-GFP-encoding recombinant retrovirus particles were harvested and used to infect HeLa cells. Selection, amplification and maintenance of the individual clones were performed in the presence of G418 (500 μg/ml; Gibco). Expression of control GFP or ORFV073-GFP was monitored by fluorescence microscopy and Western blot using antibody against GFP (sc-9996; Santa Cruz Biotechnology).
7
Autophagy Regulation by Atg17
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Yeast cells (Saccharomyces cerevisiae) were cultured in standard rich medium (YPD) or synthetic dextrose medium (SD, 0.67% yeast nitrogen base with ammonium sulfate, 2% glucose, and appropriate amino acid supplements). Autophagy was induced by shifting to nitrogen starvation medium. TN124 Atg17∷KAN cells were transformed with yCPLAC33 and wild-type and mutant ATG17-yCPLAC33 constructs, and grown to mid-log phase. Cells were then switched to nitrogen-starvation media for 3h to induce autophagy and the Pho8Δ60 assay was carried out as previously described [44 (link), 45 (link)]. Atg17-GFP expression was characterized by western blotting against GFP with Santa Cruz antibody sc9996.
8
Western Blot Antibody Validation Protocol
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Antibodies used for western blot were: mouse anti-Flag (1:5,000, F3165, Sigma), mouse anti-Myc (1:1,000 sc-40, Santa Cruz), mouse anti-HA (1:1,000, sc-7392, Santa Cruz), mouse anti-GFP (1:500, sc-9996, Santa Cruz), mouse anti-α-tubulin (1:1,000, T5168, Sigma), rabbit anti-phospho-histone H3 (Ser 10) (1:5,000, 05-817 R, Millipore), rabbit anti-BubR1 (1:5000, A300-386A, BETHYL), mouse anti-Bub1 (1:1000, B0561, Sigma), rabbit anti-Bub3 (1:1000, ab131157, Abcam), rabbit anti-CENP-E (1:5,000, a gift from Dr. Don W. Cleveland), sheep anti-HOIP and anti-HOIL-1L (1:5,000, a gift from Dr. Philip Cohen), rabbit anti-SHARPIN (1:1,000, #4444, CST), anti-linear Ub62 (link) (1:2500, a gift from Dr. Vishva M. Dixit, Genentech Inc.), rabbit anti-Ub K48 (1:1000, 05-1307, Millipore), rabbit anti-Ub K63 (1:1000, 05-1308, Millipore), rabbit anti-Ska3 (1:3000, a gift from Dr. Hongtao Yu), rabbit anti-KNL1 (1:5000, a gift from Dr. Hongtao Yu). Western blot antibody against SHARPIN of primary MEF cells was: rabbit anti-SHARPIN (1:500, Millipore). Images have been cropped for presentation (uncropped scans of the blots were shown in Supplementary Fig. 7 ).
9
Western Blot Analysis of GFP Expression
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Total cellular protein extractions were carried out as described previously[53 (link)], and quantified using Bradford reagent (Bio-Rad), according to manufacturer’s instructions. A total of 50 μg of protein per sample was run on a 4% stacking and 12% resolving, self-made gel before being transferred to a membrane as described previously[14 (link)]. Blocking, primary (1:2000 dilution of the mouse monoclonal GFP antibody, #sc-9996, Santa Cruz Biotechnology) and secondary antibody (1:2000 dilution,[14 (link)]) incubations, and membrane revelation was carried out as described previously[14 (link)].
10
Protein Extraction and Detection
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Total proteins were extracted from ventricular myocardium or cultured NRVMs with 1× sampling buffer (41 mM tris-HCl, 1.2% SDS, and 8% glycerol). Protein concentration was determined with bicinchoninic acid (BCA) reagents (Pierce Biotechnology, Rockford, IL). Equal amounts of proteins were loaded to each lane of the SDS–polyacrylamide gel and fractionated via electrophoresis, transferred to a polyvinylidene difluoride (PVDF) membrane using a Trans-Blot apparatus (Bio-Rad, Hercules, CA) and immunodetected with anti-PDE1A (1:800; SC-50480, Santa Cruz Biotechnology), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000; G8795, Sigma-Aldrich), anti-HA (1:1000; 3724, Cell Signaling), anti-GFP (1:1000; SC-9996, Santa Cruz Biotechnology), anti-tubulin (1:1000; E7-s, DSHB), anti-CryAB (1:1000; ADI-SPA-222, Enzo Life Sciences), anti-Psmb5 [1:1000; custom-made (30 (link))], or anti–p-Rpn6 [1:5000; custom-made (13 )] as the primary antibodies and appropriate horse radish peroxidase–conjugated secondary antibodies. The bound secondary antibodies were detected using the enhanced chemiluminescence detection reagents (GE Healthcare, Piscataway, NJ). Blots were imaged and quantified using the Quantity One or Image Lab software (Bio-Rad). For some of the blots, the total protein content derived from the stain-free protein imaging technology was used as in-lane loading control (8 (link)).
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