Radio-Immunoprecipitation lysis (BB-3209, BestBio, Shanghai, China) was used to isolate the total protein from the cells. The protein was separated by SDS-PAGE and transferred onto the polyvinylidene fluoride membrane. The membrane was allowed to probe with primary antibodies including rabbit monoclonal antibodies to proliferating cell nuclear antigen (PCNA) (1:1,000, ab92552, Abcam Inc., Cambridge, UK), Ki-67 (1:5,000, ab92742, Abcam Inc.), matrix metalloproteinase (MMP)-2 (1:1,000, ab92536, Abcam Inc.), MMP-9 (1:1,000, ab38898, Abcam Inc.), Cyclin D1 (1 : 200, ab16663, Abcam Inc.), and Cyclin-dependent kinase 4 (CDK4) (1:1,000, ab108357, Abcam Inc.). Horseradish peroxidase-labeled goat anti-rabbit IgG secondary antibody (1:1,000, Wuhan Boster Biological Technology Co., Ltd., Wuhan, Hubei, China) was added and incubated with the membrane. Target protein relative expression = gray value of the target protein band/gray value of GAPDH.
Ab108357
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab108357 is a lab equipment product from Abcam. It is a device used for laboratory applications. The core function of this product is to perform specific tasks in a research or experimental setting.
Automatically generated - may contain errors
Lab products found in correlation
Ab16663, by Abcam (15 mentions)
Ab134175, by Abcam (11 mentions)
Ab32503, by Abcam (10 mentions)
Ab124821, by Abcam (9 mentions)
Pvdf membrane, by Merck Group (9 mentions)
Ab109520, by Abcam (7 mentions)
Ab181602, by Abcam (7 mentions)
Ab32147, by Abcam (6 mentions)
Ab8226, by Abcam (6 mentions)
Ab32124, by Abcam (6 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Ab92536, by Abcam (5 mentions)
Ab8245, by Abcam (5 mentions)
Ab32053, by Abcam (5 mentions)
Ab8227, by Abcam (4 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Ab76003, by Abcam (4 mentions)
Ab33911, by Abcam (4 mentions)
Ab38898, by Abcam (3 mentions)
Ab181591, by Abcam (3 mentions)
48 protocols using ab108357
1
Western Blot Analysis of Cell Signaling Proteins
2
Western Blot Analysis of Cell Signaling
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PBS at 4 °C was used to wash the cells twice. Thereafter, protease inhibitors in cold RIPA buffer were used to lyse the cells. Protein concentration was determined using BCA Protein Quantitation Assay (KeyGen Biotech, Nanjing, China). Total protein of the cells were denatured using 10% SDS-PAGE and then transferred onto a nitrocellulose membrane. 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) was used to block the membrane for 1 h at room temperature. The membranes were then incubated with the primary antibodies overnight at 4 °C (dilution ratio 1:2000). TBST was used to wash the membranes three times and they were subsequently incubated with secondary antibodies (anti-rabbit IgG or anti-mouse IgG) for 1 h at room temperature. TBST was once again used to wash the membranes three times. The targeted proteins were identified using the ECL (EMD Millipore, MA, USA) method. SKA3, CCNE2, CCNA2, CDK4, CDK2, P53, P53-pSer15, P53-pSer46 antibodies used for western blot in this research were purchased from Bioss (bs-7848R), Abcam (ab32147), Abcam (ab108357), Abcam (ab181591), Abcam (ab32103), Abcam (ab32389), CST #9284 and CST#2521.
3
Protein Expression Analysis by Western Blot
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The tissues and cells were lysed with RIPA buffer supplemented with phenlymethanesulfonyl fluoride (PMSF). After the concentration was quantified, the proteins were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were immunoblotted with the primary antibodies: CDK4 (1:500, ab108357; Abcam, Cambridge, UK), BCAS2 (1:500, ab151293; Abcam), GAPDH (1:500, ab8245; Abcam), p21 (1:500, ab109520; Abcam) and caspase-3 (1:300, ab2171; Abcam) overnight at 4°C. After being rinsed with TBST, the membranes were incubated with secondary antibodies (at a dilution of 1:5,000) conjugated to horseradish peroxidase. The protein bands were visualized and exposed to X-ray film. GAPDH was used as the internal control.
4
Molecular Analysis of ASPM in Cell Proliferation
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Anti-ASPM (For immunohistochemical analysis, 1:200 dilution, ABIN5913131. For immunoblot, 1:1000 dilution, ABIN960544, Abcam), Anti-β-actin (1:1000 dilution, ab8227, Abcam). Anti-CDK4 (1:1000 dilution for immunoblot, 1:200 dilution for immunohistochemical analysis, ab108357, Abcam), Anti-CCND1 CCND1 (1:10000 dilution, ab134175, Abcam).
The quantitative PCR primer sequences of ASPM were as follows:
The quantitative PCR primer sequences of ASPM were as follows:
Forward, 5'-GGGAAAGGCAAATGGAAAAC-3'; and,
Reverse, 5'- CCCAAGGCCATACAAGTGTT-3'.
Forward, 5'-CGACCACTTTGTCAAGCTCA-3'; and,
Reverse, 5'-GGTTGAGCACAGGGTACTTTATT-3'.
5
Immunoblotting Analysis of Cell Cycle Regulators
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6
Protein Expression Analysis in Clinical Samples
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The RIPA lysis buffer (Beyotime, China) was used extract total proteins from clinical tissues and cell lines. The proteins were separated by 10% sodium sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the targeted protein bands were transferred onto PVDF membrane (Sigma, USA). Subsequently, the PVDF membranes were probed with the primary antibodies including anti-β-actin (#ab8226, Abcam, USA), anti-SOD2 (#ab13534, Abcam, USA), anti-NLRP3 (#ab214185, Abcam, USA), anti-CyclinD1 (#ab16663, Abcam, USA), anti-Cyclin E2 (#ab32103, Abcam, USA), anti-CDK2 (#ab32147, Abcam, USA), anti-CDK4 (#ab108357, Abcam, USA), anti-CDK6 (#ab124821, Abcam, USA), anti-Caspase 3 (#ab13847, Abcam, USA), anti-Bax (#ab32503, Abcam, USA), anti-Bcl-2 (#ab185002, Abcam, USA), anti-Caspase 1 (#ab238979, Abcam, USA) and anti-IL-1β (#ab33774, Abcam, USA). After that, the membranes were incubated with anti-Rabbit IgG antibody at 4°C for 24h, and the enhanced chemiluminescent (ECL) system was employed to detect the protein bands. Finally, all the protein bands were quantified by Image J software. The primers sequences of the target genes were listed in Table 1 .
7
Western Blot Analysis of Cell Signaling
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P19G1-treated cells were collected and washed with ice-chilled DPBS. Cells were
lysed with radioimmunoprecipitation assay (RIPA) reagent supplemented with
protease and phosphatase inhibitors (Transgen Biotech, Beijing, China) according
to the manufacturer's instructions. Protein concentrations were determined with
the Enhanced BCA Protein Kit (Beyotime Biotechnology). The protein samples were
then subjected to western blot analysis as described previously.21 (link)Antibodies against CDK-4 (ab108357, 1:1000), CDK-6 (ab124821, 1:1000), MMP-2
(ab92536, 1:1000), and MMP-9 (ab76003, 1:1000) were purchased from Abcam
(Abcam). Antibodies against BCL-2 (15071T, 1:1000), Caspase-3 (9662S, 1:1000),
and Cleave-caspase 3 (9661T, 1:1000) were purchased from Cell Signaling
Technology (Danvers, MA, USA), and antibodies against β-actin (GB15001, 1:1000)
were purchased from Servicebio (Servicebio, Wuhan, China). Detection was
analyzed by the Azure Biosystem25 (link) (Azure c600, Azure
Biosystem™).
lysed with radioimmunoprecipitation assay (RIPA) reagent supplemented with
protease and phosphatase inhibitors (Transgen Biotech, Beijing, China) according
to the manufacturer's instructions. Protein concentrations were determined with
the Enhanced BCA Protein Kit (Beyotime Biotechnology). The protein samples were
then subjected to western blot analysis as described previously.21 (link)Antibodies against CDK-4 (ab108357, 1:1000), CDK-6 (ab124821, 1:1000), MMP-2
(ab92536, 1:1000), and MMP-9 (ab76003, 1:1000) were purchased from Abcam
(Abcam). Antibodies against BCL-2 (15071T, 1:1000), Caspase-3 (9662S, 1:1000),
and Cleave-caspase 3 (9661T, 1:1000) were purchased from Cell Signaling
Technology (Danvers, MA, USA), and antibodies against β-actin (GB15001, 1:1000)
were purchased from Servicebio (Servicebio, Wuhan, China). Detection was
analyzed by the Azure Biosystem25 (link) (Azure c600, Azure
Biosystem™).
8
Immunofluorescence Staining of CDK4 and pRb
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For immunofluorescence staining, A549 and PC9 cells were seeded on 10 mm confocal dishes and treated with 0.1 μmol/L pemetrexed and/or 10 μmol/L ribociclib. Equal amounts of DMSO were added to control cells. After 72h, cells were fixed in 4% PFA for 30 min, permeabilized in 0.3% Triton X-100 for 20 min, and blocked in 5% normal goat serum for 60 min at room temperature. Sequentially, cells were incubated with primary antibody against CDK4 (1:200 dilution, #ab108357, Abcam, USA) or phosphor-Rb (1:200, #8516T, Cell signalling Technology, USA) at 4°C overnight, then washed with phosphate buffered-saline with Tween-20 (PBST) and incubated with FITC-labelled secondary antibody (1:100 dilution, Proteintech, Wuhan, China) for 1 h at room temperature. The nuclei were labelled using 4′,6-diamidino-2-phenylindole (DAPI) (2 mg/mL) in the dark for 15 min, and imaging was performed on a fluorescence microscope (Olympus IX 73 DP80, Japan).
9
Western Blot Analysis of BC Proteins
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Western blot analysis was executed as previously described [23 (link)]. BC tissues and cells were lysed in lysis buffer (Beyotime). The bands were assessed through EZ-ECL chemiluminescence (Biological Industries, Beit-Haemek, Israel) and visualized using an ImmunoStar LD (Wako Pure Chemical, Osaka, Japan). The primary antibodies were as follows: anti-cyclin D1 (ab16663, 1:1,000; Abcam, Cambridge, MA, USA), anti-Cell Cycle Dependent Kinase 4 (CDK4) (ab108357, 1:500; Abcam), anti-GAPDH (ab128915, 1:5,000; Abcam), and anti-SCUBE2 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Goat anti-rabbit (ab97051, 1:10,000; Abcam) or mouse (ab205719, 1:2,000; Abcam) immunoglobulin G (IgG) was used as the secondary antibody. GAPDH was regarded as a loading control.
10
Western Blot Analysis of Cell Cycle Regulators
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For western blot analysis, cells or tumor tissue were lysed in lysis buffer from the Qproteome Mammalian Protein Prep Kit (37901, QIAGEN) with the addition of protease inhibitors after PBS washing. Protein concentrations were measured by a microplate reader (Molecular Devices, California, USA) using the BCA Protein Assay Kit (P0010S, Beyotime, China). Then equal amounts of protein were resolved on SDS-PAGE and transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA) and incubated with primary antibodies against: CDK4 (1:5000, ab108357, Abcam), cyclin D1 (1:1000, 554180, BD Bioscience, USA), cyclin A2 (1:2000, ab181591, Abcam), cyclin B1 (1:50000, ab32053, Abcam), P21 (1:5000, ab109520, Abcam), PD-L1 (1:500, ab205921, Abcam or 1:2000, PA5-28115, Thermo Fisher scientific) and β-actin (1:2000, ab8227, Abcam); Secondary antibodies were goat anti-rabbit HRP (1:10000, ab6721, Abcam) and goat anti-mouse HRP (1:5000, ab97023, Abcam). Immunoreactive polypeptides were detected by electrochemiluminescence (ECL) reagents (Cat#170-5061, Bio Rad) using ChemiDoc™ XRS + Imaging System (Bio-Rad). Western blot band intensity quantification was calculated using ImageJ.
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