The treated cells were lysed with radio immunoprecipitation assay (RIPA) buffer (Beyotime, Nanjing, China) on ice. The supernatant of the lysis was collected after centrifugation for 15 min at 12,000 g and 4 °C. The protein concentrations were tested using the Bradford protein assay kit (Beyotime, Shanghai, China). Each 30 µg protein was added with loading buffer (Beyotime) and was heated to 100 °C for 10 min. The samples were separated with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels (Beyotime, Shanghai, China), transferred to a 0.22 µm polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Darmstadt, Germany), incubated with primary (anti-RGS1, ab154973, 1:500; anti-β-actin, ab8227, 1:3,000. Abcam, London, UK) and secondary (goat anti-rabbit immunoglobulin G (IgG), ab97051, 1:5,000, Abcam) antibodies, and visualized under a ChemiDoc XRS+ detector with chemiluminescent substrate ECL kit (Merck Millipore) according to the manufacturer’s instructions. Finally, the visualized lanes were analyzed with Image J software (National Institutes of Health, USA).
Ab8227
Manufactured by Merck Group
Sourced in Germany, United States
Ab8227 is a laboratory equipment product. It is designed for general laboratory use. The core function of Ab8227 is to provide a reliable and consistent tool for laboratory tasks.
Automatically generated - may contain errors
Lab products found in correlation
Ab97051, by Abcam (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Bradford protein assay kit, by Beyotime (1 mentions)
Loading buffer, by Beyotime (1 mentions)
Ab97080, by Abcam (1 mentions)
C2206, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Tgx precast gel, by Bio-Rad (1 mentions)
Ecl based detection, by Thermo Fisher Scientific (1 mentions)
Radioimmunoprecipitation assay (ripa), by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Ab61682, by Abcam (1 mentions)
Ab110413, by Abcam (1 mentions)
Ab10983, by Abcam (1 mentions)
Chemidoc molecular imager, by Bio-Rad (1 mentions)
Sc 11415, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
10 protocols using ab8227
1
Western Blot Analysis of RGS1 Protein
2
Western Blot Analysis of Protein Expression
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3
Western Blot Analysis of Embryonic Brain and Eye
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Brains and eyes were harvested from Stages 40/41 embryos that were anesthetized with 0.4mg/ml MS222 in 1X MBS. The tissues were mechanically homogenized with radioimmunoprecipitation assay (RIPA; Sigma) buffer supplemented with protease inhibitor cocktail (Roche) by repeated pipetting, and solubilized by constant rotation at 4°C for 30 min. Remaining non-solubilized material was then pelleted by centrifugation at 13000rpm for 10 min and only the supernatant was then retained. Protein concentration was determined by Bradford assay (Bio-Rad) and spectrophotometry. Bovine serum albumin (BSA; Invitrogen) was used to create a standard curve for protein concentration and for normalizing the concentration between samples. The lysates were resolved by 12% TGX precast gels (BioRad) at constant 20mA, transferred to nitrocellulose membrane (BioRad) at constant 110V and subjected to western blot analysis by incubating with a rabbit anti-β-actin (Abcam Cat# ab8227, RRID:AB_2305186 ; 1:8000) or rabbit anti-β-catenin (Sigma-Aldrich Cat# C2206, RRID:AB_476831 ; 1:8000) antibody at 4°C for overnight. The blots were then incubated with HRP-conjugated secondary antibodies (Abcam Cat# ab97080, RRID:AB_10679808 ; 1:16000) in room temperature for one hour, followed by ECL-based detection (Invitrogen).
4
Western Blotting for Mitochondrial Proteins
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Proteins were transferred to methanol-activated PVDF membrane in xCell SureLock Mini-Cells under 30 V constant voltage for 1 h at 4 °C. Coomassie was washed off of blue native blots using 100 % methanol. Blots were blocked with 3 % BSA in PBST (1 mL/L Tween-20/PBS) and incubated with primary antibody diluted in 1 % BSA/PBST overnight at 4 °C. The next day, blots were washed 3 × 10 min in PBS-Tween, probed with Alexa Fluor 488, IRDye 800CW, or IRDye 680RD conjugated secondary antibodies diluted in blocking solution for 1 h at room temperature, and rinsed again 3 × 10 min in PBST. Detection was achieved using ChemiDoc Molecular Imager (BioRad). Band densitometry was quantified using ImageJ Gel Plugin (NIH). We used the following antibodies: mtOXPHOS cocktail for combined ATP5A, UQCRC2, MTCO1, SDHB, and NUDFB8 detection(Abcam, ab110413), PLIN1 (Abcam, ab61682), UCP1 (Abcam, ab10983), β-actin (Abcam, ab8227), vinculin (Sigma-Aldrich, V9131), and TOMM20 (Santa Cruz Biotechnology, sc-11415).
5
Western Blot Analysis of PKA and cAMP in HCM Cells
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HCM cells were collected and treated with 1X SDS cell lysis buffer (Beyotime Inc., Shanghai, China). After SDS-PAGE, the proteins were transferred to the PVDF membrane (Millipore, Shanghai, China) at 110 V for 90 min. The membranes were blocked at 37°C for 100 min and then incubated with polyclonal goat anti-human PKA (R&D Systems, Inc., Minneapolis, MN, USA cat no. AF4177, 1:500 dilution) and monoclonal mouse anti-human cAMP antibody (R&D Systems, Inc., Minneapolis, MN, USA, cat no. MAB2146, 1:500 dilution) at 4°C overnight. Subsequently, the membranes were rinsed and incubated with anti-goat secondary (PKA, cat. no. ab339770) and HRP-mouse secondary (cAMP, cat. no. ab157532) antibodies (1:2,000; Abcam, Cambridge, MA, USA) at 37°C for 30 min. The reference protein used in this study was rabbit polyclonal to human β-actin (ab8227, 1:500) (Sigma, St. Louis, MO, USA).
6
CASK Protein Quantification by Simple-Western
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Cells were dissociated in extraction buffer (50 mM Tris-HCl, 100 mM NaCl, 5 mM EDTA, and 1 mM EGTA) using plastic cell scraper and lysed with 6 short sonication bursts at 36% amplitude (Vibra-Cell VCX-600, Sonics). About 250 µg/ml protein was loaded for Simple-Western WES (ProteinSimple) quantification with antibodies for CASK (1:500 Novus Biologicals NBP2-41181), beta-actin (1:100 Abcam ab8227), and GAPDH (1:5000 Sigma G9545). Compass Software For Simple Western (Version 4.0.0) was used to identify protein-specific peaks. In the chromatogram, the peak area was used for protein quantification. We normalized CASK protein for housekeeping protein. For time point days 0, 8, and 16, we obtained two and three biological replicates for time point 28 and siRNA-mediated knockdown of CASK. Biological replicates were seeded at different dates, using different passages. Statistical significance between cell lines was determined with ANOVA and post hoc Tukey HSD in R.
7
Western Blot Analysis of Cellular Proteins
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Protein extracts were prepared in Laemmli loading buffer containing 2.5% beta-mercaptoethanol (except for experiment in non-reducing condition where no beta-mercaptoethanol was added) and heated for 5 min at 95°C before separation of 20 μg total proteins on 8 or 12% SDS-PAGE gels. Separated proteins were transferred onto nitrocellulose or PVDF membranes. The membranes were blocked in TBS (Tris-buffered saline; 50 mM Tris-Cl, pH 7.6, 150 mM NaCl), 0.1% Tween-20, 5% non-fat milk. The following antibodies were used: anti-GFP (Santa Cruz sc-9996, 1:1,000), anti-GABARAP/Atg8a (Cell Signaling Technology No. 13733, 1:2,000), anti-Ref(2)P (Abcam ab178440, 1:1,000), anti-β actin (Abcam ab8227, 1:2,000), anti-α tubulin (Sigma-Aldrich T5168, 1:40,000), HRP-coupled secondary antibodies anti-rabbit and anti-mouse (Thermo Scientific No. 31460 and 31450, 1:10,000). Signals were developed using the ECL detection reagents (Amersham, RPN2209).
8
Western Blot Analysis of Purine Metabolism Enzymes
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Cells were digested with Accutase (Gibco, Carlsbad, CA, USA) and lysed in TNTE buffer with protease inhibitors (Sigma, St. Louis, MO, USA). Subsequently, the lysates were subjected to SDS-PAGE, and the proteins were transferred to nitrocellulose membranes. The following primary antibodies were used: anti-Nestin (1:500, ABclonal, A11861), anti-SOX2 (1:3000, Cell Signaling Technology, 5067S), anti-H6PD (1:500, Sigma, A06407), anti-IMPDH1 (1:1000, Abcam, ab33039), anti-PPAT (1:500, ORIGENE, TA504559), anti-G6PD (1:1000, Hangzhou HuaAn Biology, R1706-7), anti-ADSS (1:500, ABclonal, A3720), anti-IMPDH2 (1:2000, Proteintech, 12948-1-AP), anti-ADSL (1:1000, Abcam, ab154182), anti-HPRT (1:500, Abcam, ab10479), anti-APRT (1:400, ABclonal, A5456), and anti-β-actin (1:5000, Sigma, ab8227) as an internal control. The primary antibodies were revealed using the appropriate secondary antibody conjugated to peroxidase and enhanced chemiluminescence (Thermo Fisher Scientific, Waltham, MA, USA).
9
Analyzing Protein Expression in Hippocampi
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Protein samples were isolated from hippocampi or cells using RIPA lysis buffer (150 mM NaCl, 1% Triton™ X-100, 0.5% sodium deoxycholate, 0.1% SDS and 50 mM Tris pH8.0) supplemented with PhosSTOP™ and protease inhibitors (Roche). Protein levels were determined by resolving 20 μg of protein on SDS-PAGE gels, transferred onto nitrocellulose or PVDF membranes and incubated with primary antibodies for eIF2α-P (1:1000; Cell Signaling 3597s), eIF2α (1:1000; Cell Signaling 2103s), ATF4 (CREB-2, 1:1000; Santa Cruz sc200), ATF6 (1:1000; Genetek 70B1413), GSK3β (1:2000, Cell Signaling, 9832), pSer9-GSK3β (1:1000, Cell Signaling, 9322), total tau (tau-5, 1:2000; Invitrogen ANB0042), p-tau (AT100, 1:2000; Thermo Fisher Scientific MN1060). Horseradish peroxidase-conjugated secondary antibodies (1:5000; Dako) were applied and protein visualized using enhanced chemiluminescence (GE Healthcare) and quantitated using ImageJ. Antibodies against GAPDH (1:5000; Santa Cruz sc32233), β-actin (1:5000; Abcam ab8227) and β-tubulin (1:5000; Millipore MAB1637) were used to determine loading. To detect PrPSc (prion protein) homogenized samples were digested with 50 μg/ml of proteinase K (PK) at 37°C for 1 h prior to electrophoresis. Membranes were then probed with ICSM-35 (1:10 000; D-GEN 0130-03501) and goat anti-mouse (1:10 000; Dako).
10
Western Blot Analysis of Tau Protein
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TBS-soluble samples were incubated with reducing 6× Laemmli SDS buffer (Alfa Aesar, J61337) at 95 °C for 10 min for denaturing conditions or 4× NuPAGE sample buffer (Invitrogen NP0007) for non-denaturing conditions and then loaded on 4–12% NuPAGE Novex gels (Invitrogen). Nitrocellulose membranes were used to transfer proteins and blocked with 5% BSA in TBS with 0.01% tween followed by overnight incubation of primary antibodies (HT7 (Thermo Fisher, MN1000; 1:1,000 dilution), anti-Bassoon (Millipore, ABN255; 1:1,000 dilution), MC1 (Peter Davies; 1:1,000 dilution), anti-total tau (DAKO, A0024; 1:5,000 dilution), PHF1 (Peter Davies; 1:1,000 dilution), anti-Actin (Abcam, ab8227; 1:2,000 dilution), pThr231 (Millipore, MAB5450; 1:1,000 dilution) and Vinculin (Sigma, V9131; 1:1,000 dilution)) diluted in the blocking solution. Horseradish peroxidase (HRP) secondary antibodies (goat anti-mouse HRP conjugated (Invitrogen, 626820; 1:5,000 dilution) and goat anti-rabbit HRP conjugated (Invitrogen, 31460; 1:5,000 dilution)) were incubated for 1 h at RT and the proteins were detected with Supersignal West Pico (Thermo Scientific, 34580) and imaged by using iBright 1500 (Invitrogen). Western blots were analyzed using ImageJ (NIH, v1.53i).
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