We followed a method in ref. 23 (link). Briefly, human embryonic kidney (HEK293T) cells in 12-well plates were co-transfected with pcDNA3.1-based A3G, sA3G, or their mutant expression vector (0.66 μg) and pcDNA-HVif, pcDNA-HVif-H41A/H42A or empty pcDNA3.1 vector (1.32 μg) with the transfection reagent, FuGene HD (Promega). After incubation for 24 h, 2 μM MG132 or DMSO was added to the medium, and cells were incubated for an additional 24 h. Resulting cell lysates were analyzed by SDS-PAGE and immunoprobed with monoclonal anti-c-myc (9E11, Abcam), anti-Vif (319, Abcam), or anti-α-tubulin primary antibodies (DM1A, Abcam), and monoclonal anti-mouse IgG secondary antibody conjugated with a fluorophore (ab216772, Abcam). Protein bands were detected with a ChemiDoc system (Bio-Rad Laboratories) and analyzed using NIH ImageJ59 (link).
Ab216772
Manufactured by Abcam
Sourced in United States
Ab216772 is a biotechnology product that can be used for laboratory research purposes. It is a tool that researchers can utilize in their experiments, but no further details about its core function or intended use can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Ab216773, by Abcam (3 mentions)
Ab8226, by Abcam (3 mentions)
Irdye 800cw, by Abcam (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Fugene hd, by Promega (1 mentions)
Chemidoc system, by Bio-Rad (1 mentions)
Ab129002, by Abcam (1 mentions)
Free protease inhibitor cocktail tablet, by Roche (1 mentions)
Ab216777, by Abcam (1 mentions)
Gtx125926, by GeneTex (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
C43 ab128193, by Abcam (1 mentions)
Irdye 680rd, by Abcam (1 mentions)
F1804, by Merck Group (1 mentions)
Ab108349, by Abcam (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Ab192890, by Abcam (1 mentions)
Ab109012, by Abcam (1 mentions)
Ripa lysis buffer, by Cell Signaling Technology (1 mentions)
6 protocols using ab216772
1
Investigating APOBEC3G Regulation by HIV-1 Vif
2
Visualization of Protein Expression in Mini-Genome Assays
3
Western Blot Analysis of Cell Lysates
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Cells (~5 × 106) were lysed in RIPA buffer RIPA Buffer (50 mM Tris-HCl, pH 8.0, with 150 mM sodium chloride, 1.0% Igepal CA-630 (NP-40), 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) supplemented with complete protease inhibitor cocktail, kept on ice for 20 min. Lysates were then cleared by centrifugation (12,300 × g, 10 min, 4 °C). Protein concentrations in the cleared supernatants were determined with Bio-Rad protein assay (Bio-Rad, CA) according to the manufacturer’s instructions. In total, 20 µg proteins were separated by SDS–PAGE and transferred to nitrocellulose membranes. Membranes were blocked and incubated with antibodies against vinculin (1:40,000, MAB3574 millipore Sigma), CDKN2A/p16INK4A [EPR1473] (1:500, ab108349, Abcam), CDKN2A/p14ARF [EPR17878] (1:500, ab185650, Abcam), Primary antibodies were detected by fluorescent Goat anti Rabbit IgG H&L (1:10,000, IRDye® 800CW preadsorbed, ab216773, Abcam) and Goat anti-Mouse IgG H&L (1:10,000, IRDye® 800CW) preadsorbed ab216772, Abcam). Membranes were imaged using Odyssey CLx imaging system (Li-Cor, Nebraska, USA).
4
Western Blot Analysis of Autophagy Markers
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HPAECs were dissolved in RIPA lysis buffer (Cell Signaling Technology, Danvers, MA, USA). Protease inhibitors were added, and the lysate was centrifuged at 12,000 rpm for 15 min at 4°C. The protein concentration was determined using the bicinchoninic acid protein assay. Proteins were separated and an adequate concentration was added for SDS-PAGE, followed by electrotransfer onto polyvinylidene difluoride membranes. The membranes were blocked using 5% bovine serum albumin with 0.1% Tween 80, incubated with primary antibodies overnight at 4°C, and then washed three times with TBST. The membranes were probed with antibodies against LC3 (ab192890; 1:2000; Abcam) as an autophagy marker protein. An increase in the LC3II/LC3I ratio is indicative of autophagy activation. The membranes were also probed with antibodies against P62 (ab109012; 1:10,000; Abcam), PIF1 (sc-48,377; 1:100; SANTA CRUZ), and β-actin (ab8226; 1:1000; Abcam). Finally, the membranes were incubated with anti-rabbit (ab216773; 1:10,000; Abcam) or anti-mouse (ab216772; 1:10,000; Abcam) secondary antibodies for 1 h. Protein bands of interest were detected using an Amersham Imager 600 (Cytiva, Marlborough, MA, USA) and quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
5
Quantitative Protein Analysis by Western Blot
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Intracellular protein was extracted from cells using the NucBuster Protein Extraction Kit (Merck, 77183) and quantified using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23225). A total of 40 µg of cytoplasmic protein was diluted in laemmli buffer (Bio-Rad, 1610747) with beta-mercaptoethanol, denatured at 95 °C for 5 min and loaded onto Mini-PROTEAN TGX Gels (Bio-Rad, 4568084). Samples were transferred to nitrocellulose membranes using the TransBlot Tubro system (Bio-Rad, 170–4270) and protein visualised with Ponceau Stain (G-Biosciences). Membranes were blocked in 5% BSA TBS-T (5% BSA in 1× Tris-buffered saline and 0.1% Tween 20) for 1 h and incubated with primary antibodies overnight in 5% BSA TBS-T (MMP1 1:1000 ab137332, MMP2 1:1000 D4M2N Cell Signalling, B-actin 1:10,000 ab8226, Abcam). Membranes were washed with TBS-T and incubated in secondary antibodies (Dnk pAb to Rb IgG IRDye 680RD, ab216779, Goat pAb to Ms IgG IRDye 800CW, ab216772, Abcam) for 1 h at room temperature. Membranes were visualised using the Odyssey CLx system (Licor).
6
Immunoblotting Analysis of Tumor Samples
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The CAL27 and SCC15 cells with the indicated treatment and tumor tissues (tumor tissue was ground using a tissue grinder) from tumorigenicity analysis in the nude mice were isolated by RIPA reagents supplemented with 1% proteinase inhibitor (Sigma, USA) and quantified by BCA Kit (Sigma, USA). The samples were subjected into SDS-PAGE gel electrophoresis (12%) and transferred into PVDF membranes (Millipore, USA), blocking using nonfat milk (5%) at 25°C for 2 hours and incubating with primary antibodies overnight at 4°C. The samples were further incubated using secondary antibodies at 25°C for 1.5 hours at room temperature and detected using enhanced chemiluminescence solution (Millipore, USA). The antibodies information was shown as follows: SLC7A11 (ab175186, Abcam, USA), β-actin (ab8226, Abcam, USA), GPX4 (ab125066, Abcam, USA), second antibodies (ab216773 (Goat anti-Rabbit), ab216772 (Goat anti-Mouse), Abcam, USA).
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