To extract proteins from hASMCs, we used RIPA buffer (abs9229, absin, China). After quantification of the extracted proteins, denaturation was performed. The protein was harvested for electrophoresis. After being electroblotted onto a nitrocellulose membrane, 5% BSA was utilized to seal the immunoblot (37°C, 120 min). Afterward, primary antibodies were added (4°C, 12 h). Then, the membrane was immersed in anti-rabbit IgG H&L (1 : 1000) at 37°C for another 1 h. A color reagent (1705061, BIO-RAD, USA) was then added. The blots were observed under an Imaging System (Odyssey CLx, LI-COR, USA). The primary antibodies of Ki67 (1 : 5000), PCNA (1 : 10000), p21 (1 : 10000, ab109520), p27 (1 : 5000, ab32034), FoxO3a (1 : 10000, ab109629), and GAPDH (1 : 10000, ab181602) were bought from Abcam (UK).
Ab32034
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab32034 is a laboratory equipment product manufactured by Abcam. It is designed for use in scientific research applications. The core function of this product is to [CORE_FUNCTION_DESCRIPTION].
Automatically generated - may contain errors
Lab products found in correlation
Ab109520, by Abcam (9 mentions)
Ab134175, by Abcam (8 mentions)
Ab32503, by Abcam (7 mentions)
Ab181602, by Abcam (5 mentions)
Ab32124, by Abcam (4 mentions)
Ab16663, by Abcam (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (4 mentions)
Ab13847, by Abcam (3 mentions)
Ab108357, by Abcam (3 mentions)
Ab109199, by Abcam (3 mentions)
Ab179463, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ab109629, by Abcam (2 mentions)
Ab75814, by Abcam (2 mentions)
Ab38449, by Abcam (2 mentions)
Ab8805, by Abcam (2 mentions)
Ab2302, by Abcam (2 mentions)
Ab181861, by Abcam (2 mentions)
44 protocols using ab32034
1
Protein Extraction and Western Blot Analysis of hASMCs
2
Dexamethasone Regulation of FKBP51
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Dexamethasone (Dex) were purchased from Selleck (Selleck Chemicals, United States). The FKBP51 shRNA plasmid (h) (sc-35380-sh), scramble shRNA (sc-108060) and transfection reagent (sc-108061) as well as the primary antibodies against FKBP51 (sc-271547) and GAPDH (sc-47724) were purchased from Santa Cruz Biotechnology, Inc. (Shanghai, China). FKBP51 lentiviral expression vectors were constructed and packaged by Shanghai GeneChem BioTECH (Shanghai, China). Puromycin, Ara-C and AKT inhibitor (A6730) were purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). Primary antibodies against phospho-GSK3β (S9) (ab75814), GSK3β (ab32391), P21 (ab109520), P27 (ab32034), p-FOXO1A (S256) (ab31339), FOXO1A (ab52857), BAX (ab32503) and BCL-2 (ab32124) were purchased from Abcam (Shanghai, China). The primary antibodies against phospho-AKT (Ser473) (4060S) and AKT (pan)(4685S) were purchased from Cell Signalling Technology, Inc. (Danvers, MA, USA). The goat anti-rabbit horseradish peroxidase-labelled secondary antibody (Z2301) was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. (Beijing, China).
3
Western Blot Analysis of Cell Signaling
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Proteins were isolated with RIPA buffer (JRDUN Biotech., Shanghai, China) that contained protease and phosphatase inhibitors, which then went through 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were transferred onto nitrocellulose membranes (Millipore, Billerica, MA, USA), and blocked by 5% nonfat milk. The membranes were probed overnight at 4°C with antibodies against: NEDD4L (Ab240753, Abcam, Cambridge, MA, USA), p21 (Ab107099, Abcam), p27 (Ab32034, Abcam), ENO1 (Ab227978, Abcam) and GAPDH (#5174, Cell Signaling Technology). After wash, membranes were treated with horseradish peroxidase conjugated secondary antibody for 1 h, then with substrate (ECL; Bio-Rad, Richmond, CA, USA) followed by signal reading.
4
Protein Expression Analysis in Transfected Cells
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Transfected SGC-7901 cells were lysed in lysis buffer (Roche, Basel, Switzerland) for protein extraction at 3 days post-transfection. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Maibio, Shanghai, China). After blocking in 5% skim milk for 2 h at room temperature, the membranes were incubated with the specific primary antibodies for p27Kip1 (ab32034), p21Cip1 (ab109520), pro-CASP3 (ab2171), cleaved-CASP3 (ab2302), BCL2 (ab32124), BAX (ab32503), AKT (ab8805), and phosphorylated AKT (p-AKT, ab38449, Abcam, Cambridge, UK) at 4°C overnight. β-actin (ab8227) was used as an internal control. The membranes were washed in PBS 3 times and incubated in HRP-conjugated secondary antibodies for 1 h at room temperature. Positive signals were developed by ECL Plus Western Blotting Substrate (Thermo Scientific) and analyzed by ImageJ 1.49 (National Institutes of Health, Bethesda, MD).
5
Quantifying Spinal Cord Protein Expression
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A spinal cord segment, 0.5 cm in length, from where the injured site was centered
was used for protein quantification using western blotting. Proteins of the
nucleus and cytosol were extracted using the NE-PER Nuclear and Cytoplasmic
Extractions Kit (Thermo Fisher Scientific, Waltham, MA, USA), following the
manufacturer’s instructions. The protein mixture extracted from each segment was
separated via electrophoresis and transferred to the polyvinylidene difluoride
membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were first
incubated with PFKFB3 (1:5,000, ab181861, Abcam, Cambridge, UK), Bax (1:1,000,
ab32503, Abcam), Bcl-2 (1:500, ab59348, Abcam), cleaved caspase-3 (CC-3, 1:500,
ab13847, Abcam), p-CDK1 (1:1,000, ab201008, Abcam), p27 (1:5,000, ab32034,
Abcam), H3 (1:5,000, ab1791, Abcam), myelin basic protein (MBP) (1:1,000,
ab209328, Abcam), APP (1:1,000, ab32136, Abcam), β-actin (1:5,000, ab8226,
Abcam), and p-p27 (1:1,000, ab75908, Abcam) antibodies at 4°C overnight,
respectively, and incubated with the secondary antibodies (1:10,000, ZB-2301 or
ZB-2305, Zhongshan Gold Bridge, Beijing, China) at 25°C for 1 h. The proteins
were detected using an ECL kit (Immobilon, Millipore, Billerica, MA, USA). The
gray values of each band were calibrated according to the internal reference and
compared to those of the sham group to acquire a relative value.
was used for protein quantification using western blotting. Proteins of the
nucleus and cytosol were extracted using the NE-PER Nuclear and Cytoplasmic
Extractions Kit (Thermo Fisher Scientific, Waltham, MA, USA), following the
manufacturer’s instructions. The protein mixture extracted from each segment was
separated via electrophoresis and transferred to the polyvinylidene difluoride
membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were first
incubated with PFKFB3 (1:5,000, ab181861, Abcam, Cambridge, UK), Bax (1:1,000,
ab32503, Abcam), Bcl-2 (1:500, ab59348, Abcam), cleaved caspase-3 (CC-3, 1:500,
ab13847, Abcam), p-CDK1 (1:1,000, ab201008, Abcam), p27 (1:5,000, ab32034,
Abcam), H3 (1:5,000, ab1791, Abcam), myelin basic protein (MBP) (1:1,000,
ab209328, Abcam), APP (1:1,000, ab32136, Abcam), β-actin (1:5,000, ab8226,
Abcam), and p-p27 (1:1,000, ab75908, Abcam) antibodies at 4°C overnight,
respectively, and incubated with the secondary antibodies (1:10,000, ZB-2301 or
ZB-2305, Zhongshan Gold Bridge, Beijing, China) at 25°C for 1 h. The proteins
were detected using an ECL kit (Immobilon, Millipore, Billerica, MA, USA). The
gray values of each band were calibrated according to the internal reference and
compared to those of the sham group to acquire a relative value.
6
Antibody Detection of EGFR and p27/Kip1
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EGFR was detected with an antibody purchased from Enzo Life Sciences, Farmingdale, NY, USA (cat. ALX-804-064-C100). The p27/Kip1 antibody (ab32034) was purchased from Abcam, Cambridge, UK. Other antibodies applied in this study were anti-vinculin (V4505, Sigma) and anti-βtubulin (T4026, Sigma). Secondary antibodies were purchased from Promega (Madison, WI, USA) or Jackson Laboratories (Bar Harbor, ME, USA). PLX-4720 was purchased from Med Chem Express (Monmouth Junction, NJ, USA) and resuspended in DMSO as the vehicle. OTX-008 was purchased from Axon Medchem (Reston, VA, USA). Purified Galectin-1 (cat: 10290-HNAE) was purchased from Sino Biological Europe GmbH (Eschborn, Germany).
7
Immunoblotting Analysis of Cell Cycle Regulators
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8
CDKN1B Immunohistochemical Evaluation
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The tissue sections were incubated with CDKN1B antibody (Human & Mouse; Abcam, ab32034 & ab227911) overnight at 4°C. Next, HRP‐conjugated anti‐rabbit IgG (CST, 7074) was added and incubated with the sections for 60 minutes at 37°C. The sections were then washed three times for 5 minutes in PBS, and they were then incubated with 3,3’‐diaminobenzidine (DAB) for 30 seconds. Tissue microarrays were scanned using a Pannoramic MIDI (3D HISTECH) to acquire an immunohistochemistry (IHC) score that was used to evaluate the expression of CDKN1B.
9
Characterization of MCM Protein Expression
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For WB, the detailed procedures were performed as described previously [8] using the following primary antibodies: anti-MCM2 (Abcam, ab4461), anti-MCM3 (Abcam, ab128923), anti-MCM4 (Abcam, ab4459), anti-MCM5 (Abcam, ab75975), anti-MCM6 (Abcam, ab201683), anti-MCM7 (Abcam, ab2360), anti-CDK9 (Abcam, ab76320), anti-CyclinD1 (Abcam, ab16663), anti-CyclinE1 (Abcam, ab33911), anti-p53 (Abcam, ab241566), anti-p21 (Abcam, ab109502), anti-p27 (Abcam, ab32034), and anti-GAPDH (Abcam, ab181602).
For IHC, the standard method was described previously.14 (link) Slides were incubated with primary antibodies (Abs) against MCM2 (Immuway, YM6642), MCM3 (Abcam, ab128923), MCM4 (Immuway, YT2681), MCM5 (Abcam, ab75975), MCM6 (Abcam, ab190948), and MCM7 (Abcam, ab2360).
For IHC, the standard method was described previously.14 (link) Slides were incubated with primary antibodies (Abs) against MCM2 (Immuway, YM6642), MCM3 (Abcam, ab128923), MCM4 (Immuway, YT2681), MCM5 (Abcam, ab75975), MCM6 (Abcam, ab190948), and MCM7 (Abcam, ab2360).
10
Western Blot Analysis of DNA Damage Response
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