Kidney tissues and HUVECs were collected, and the protein concentration was determined by radioimmunoprecipitation analysis (RIPA), lysis buffer solution, and bicinchoninic acid (BCA) method. Proteins were separated by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The isolated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. The primary antibody was then incubated at 4°C overnight. The antibodies included anti-S6K (ab32529, 1:5000, Abcam, UK), anti-p-S6K (ab32529, 1:5000, Abcam, UK), anti-Beclin1 (11306-1-AP, 1: 5000, Proteintech, USA), anti-ATG5 (ab109490, 1:5000, Abcam, UK), anti-LC3 (14600-1-AP, 1: 1000, Proteintech, USA), anti-p62 (18420-1-AP, 1:2000, Proteintech, USA), anti-KLF4 (11880-1-AP, 1: 500, Proteintech, USA), anti-caspase 3 (19677-1-AP, 1:500, Proteintech, USA), anti-p-mTOR (ab109268, 1: 5000, Abcam, Cambridge, UK), anti-mTOR (ab32028, 1:2000, Abcam, UK), and anti-b-actin (60008-1-IG, 1:5000, Proteintech, USA). It was then combined with secondary anti-IgG antibodies (1:5000, SA00001-1; 1:6000, SA00001-2, Proteintech, USA). Visualization and imaging analyses were performed using chemiluminescence (Millipore, USA) and imaging software (GE Healthcare, Life Sciences, USA).
Ab32529
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab32529 is a laboratory equipment product. It is designed to serve a core function, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab109268, by Abcam (7 mentions)
Ab32028, by Abcam (5 mentions)
Ab87540, by Abcam (4 mentions)
Ab8805, by Abcam (4 mentions)
Ab134903, by Abcam (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ab81283, by Abcam (2 mentions)
Ab32047, by Abcam (2 mentions)
Ab32503, by Abcam (2 mentions)
Ab32199, by Abcam (2 mentions)
Ab207612, by Abcam (2 mentions)
Ab91109, by Abcam (2 mentions)
Anti rat cd62l, by BioLegend (2 mentions)
Anti rat cd45 pe, by Thermo Fisher Scientific (2 mentions)
Anti rat cd8a apc, by Thermo Fisher Scientific (2 mentions)
Anti rat cd44 pe, by Thermo Fisher Scientific (2 mentions)
Epics xl, by Beckman Coulter (2 mentions)
Ab59208, by Abcam (2 mentions)
Ab38449, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
20 protocols using ab32529
1
Protein Expression Analysis in Kidney and HUVEC
2
Quantifying Protein Expression in Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Patient PBMCs were lysed in RIPA buffer supplemented with cOmplete™ Protease Inhibitor Cocktail (Roche) and PhosSTOP (Roche) on ice. Protein concentration was determined using DC protein assay (Bio-Rad Laboratories) and samples prepared in 1x NuPage loading buffer (Invitrogen™) with 1x NuPage Sample Reducing Agent (Invitrogen™). Samples were run on NuPage Bis-Tris 4-12% (Invitrogen™) and NuPage Tris-Acetate 3–8% (Invitrogen™) gels and transferred using iBlot dry transfer system (Invitrogen™). Membranes were blocked 1h at RT using 5% bovine serum albumin or milk in PBSt 0.1% Tween-20 and primary antibody incubated over-night at 4°C, ENO1 (Abcam, #ab155102), ENO3 (Abcam, #ab126259), HIF-1α (BdBioscience, #610959), Akt (Abcam, #ab2771), Akt(S473) (Abcam, #ab81283), mTOR (Abcam, #ab32028), mTOR(S2448) (Abcam, #ab109268), S6K1 (Abcam, #ab32529), S6K1(T389 + T412) (Abcam, #ab60948), 4EBP1 (Abcam, #ab32024), 4EBP1(T37) (Abcam, #ab75767), or β-Actin (Sigma-Aldrich, #A5441). The secondary antibody (Dako, Aglient) was incubated 1h at RT prior detection using Amersham ECL/ECL select (GE Healthcare). Relative protein quantification was analysed using ImageLab version 6.0.1 (Bio-Rad Laboratories), results analysed using Mann-Whitney U-test or unpaired t-test and visualized using Prism 8.4.3 (GraphPad Software) (significance level, p<0.05).
3
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted from tissue samples according to the manufacturer's protocol in HGC-27 cells using the Total Protein Extraction kit (BestBio, Co., Shanghai, China) and were separated on 10% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes, which were subsequently blocked with 5% skimmed milk for 1 h at 37°C. Membranes were incubated overnight at 4°C with rabbit antibodies specific for phosphorylated (p)-S6K (1:1,000; ab32529; Abcam), β-actin (1:5,000; ab6276; Abcam), anti-p-protein kinase B (AKT; 1:1,000; 9271; Cell Signaling Technology, Inc.), anti-AKT (1:1,000; 9272; Cell Signaling Technology, Inc.), anti-mammalian target of rapamycin (mTOR; 1:1,000; 2972; Cell Signaling Technology, Inc.) or anti-p-mTOR (1:1,000; 5536; Cell Signaling Technology, Inc.). Following washing, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:3,000; ab6721; Abcam). Membranes were washed, incubated for 2 h at room temperature with an enhanced chemiluminescence substrate (Abcam) and analyzed. To quantify, signal intensities of specific bands were measured using Image Lab 3.0 software (Bio-Rad Laboratories, Inc.).
4
Western Blotting for Cellular Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was performed with the SDS-PAGE electrophoresis system. Adherent cells or adipose tissue extracts were prepared and transferred to PVDF membranes. The following primary antibodies were used: anti-GAPDH (ap0063, Bioworld), anti-Col15α1 (ab150463, Abcam), anti-Caspase-9 (ab32539, Abcam), anti-Cleaved Caspase-9 (bs7070, Bioworld), anti-Caspase-3 (Bs6428, Bioworld), anti-Cleaved Caspase-3 (bs7004, Bioworld), anti-Bcl2 (bs1511, Bioworld), anti-Bax (ab32503, Abcam), anti-AMPK (ab32047, Abcam), anti-pAMPK (ab133448, Abcam), anti-mTOR (ab87540, Abcam), anti-pmTORC1Ser2448 (ab109268, Abcam), anti-Akt (ab8805, Abcam), anti-pAktSer473 (ab18206, Abcam), anti-S6K1 (ab32529, Abcam), anti-pERK1/2 (ab201015, Abcam), anti-ERK1/2 (ab17942, Abcam), anti-pS6K1Thr389 (ab2571, Abcam), anti-Collagen I (ab34710, Abcam), anti-Collagen VI (ab6588, Abcam), anti-Fibronectin (ab2413, Abcam), anti-MMP2 (ab92536, Abcam), anti-MMP9 (ab38898, Abcam), anti-TIMP1 (WL02342, Wanleibio), anti-TIMP2 (ab180630, Abcam), anti-FGFR1 (ab31324, Abcam), anti-pFGFR1Tyr653/Tyr654 (GTX133526, GeneTex), anti-TGFβ1 (WL03092, Wanleibio). Horseradish peroxidase anti-rabbit or anti-goat (Sigma–Aldrich) were used as secondary antibodies.
5
Protein Expression Analysis in BEAS-2B Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To investigate the protein levels including ARHGEF12 (Rabbit anti-ARHGEF12 antibody, Affinity, DF4432), BCAT1 (Rabbit anti-BCAT1, ab197941, Abcam), RhoA (Rabbit Anti-RhoA), ROCK1 (RabbitAnti-ROCK1, ab134181, Abcam), PTEN (Rabbit Anti-PTEN, ab32199, Abcam), mTOR (Rabbit Anti-mTOR, ab134903, Abcam), and pS6K1 (Rabbit Anti-pS6K1, ab32529, Abcam), BEAS-2B cells treated with GS were then transfected with miRNA. After 48 h, Western Blot was employed to detect protein expression. SDS-PAGE gels electrophoresis was used to separate 20 μg total cell proteins, which were transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% milk at 4 °C overnight and then incubated with respective primary antibodies at different dilutions (ARHGEF12, 1:1000; BCAT1, 1:500; RhoA, 1:3000; ROCK1, PTEN, mTOR, pS6K1, 1:2000; GAPDH, 1:4500) at RT for 4 h. Afterwards, the membranes were further incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:6000, Millipore, Billerica, MA) at RT for 1 hr. After TBST washing, immunoreactive signals were detected using an enhanced chemiluminescence reaction (Thermo Fisher Scientific, Inc.) and recorded by an AutoChemi Imaging System (UVP LLC, CA, USA). The gray value of the bands was analyzed using MCID Elite software (InterFocus Imaging Ltd., Linton, UK).
6
Autophagy and mTOR Signaling Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Measurement of autophagy components (Beclin-1, LC3-Ⅰ/LC3-Ⅱ, and p62) and mTOR signaling (mTOR, p-mTOR, p70S6K, p-p70S6K) protein expressions in the hypothalamus by WB. Total protein concentration was measured with the BCA protein quantification kit (Beyotime, China). Samples were separated by electrophoresis, transferred to a PVDF membrane (HyBond, United States), and placed in 5% skimmed milk. The protein bands were incubated with the following primary antibodies: Beclin-1 (ab207612, 1:2000, Abcam), LC3B (3868S, 1:1000, CST), SQSTM1/p62 (ab109012, 1:10000, Abcam), mTOR (ab134903, 1:10000, Abcam), p-mTOR (5536S, 1:1000, CST), p70S6K (ab32529, 1:5000, Abcam), and p-p70S6K (9234S, 1:1000, CST). The membrane was incubated with the secondary antibodies anti-mouse IgG (H + L) (A0208, 1:1000, Beyotime) or anti-rabbit IgG (H + L) (A0216, 1:1000, Beyotime) after washing. Relative expressions of autophagy-related components and mTOR signaling were obtained by normalization against GAPDH levels.
7
Immunofluorescence Assay for Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The immunofluorescence assay (IFA) was performed as previously described (Jing et al., 2015 (link); Xiong et al., 2017 (link)). Briefly, sections were processed with relevant primary antibodies, including Bax (diluted 1:50, catalog no. ab32503, Abcam, Cambridge, United Kingdom), Bcl2 (diluted 1:50, catalog no. ab32124, Abcam, Cambridge, United Kingdom), cleaved caspase-3 (diluted 1:50, catalog no. ab13847, Abcam, Cambridge, United Kingdom), LC3B (diluted 1:50, catalog no. ab48394, Abcam, Cambridge, United Kingdom), Beclin 1 (diluted 1:50, catalog no. ab207612, Abcam, Cambridge, United Kingdom), mTOR (diluted 1:50, catalog no. ab2732, Abcam, Cambridge, United Kingdom), AKT (diluted 1:50, catalog no. ab8805, Abcam, Cambridge, United Kingdom), S6K1 (diluted 1:50, catalog no. ab32529, Abcam, Cambridge, United Kingdom), and 4E-BP1 (diluted 1:50, catalog no. ab2606, Abcam, Cambridge, United Kingdom). The sections were immersed in blocking solution and then incubated with fluorescent isothiocyanate-labeled secondary antibodies for 30 min at 37°C. After washing with PBS, the sections were stained with DAPI (catalog no. D1306, Thermo Fisher Scientific, MMAS, United States) for 10 min at room temperature and washed again with PBS. The sections were sealed with neutral resin and observed under a fluorescence microscope.
8
Isolation and Characterization of Rat CD8+ Tem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mono-nuclear cells (PBMC) were isolated from blood samples and counted by flow cytometry. Cells were then labeled with the following fluorescence-conjugated monoclonal antibodies: anti-Rat CD45 PE (12-0451-81, eBioscience, San Diego, CA, USA), anti-Rat CD8a APC (17-0081-81, eBioscience), anti-Rat CD44 PE (25-0441-81, eBioscience), anti-Rat CD62L (104432, Biolegend). CD8+Tem cells were sorted by flow cytometry (EPICS-XL, Beckman-Coulter, USA), then stained for IFN-γ (11-7311-81, eBioscience), mTOR (ab87540, Abcam, Cambridge, MA), S6K (ab32529, Abcam), T-bet (ab91109, Abcam), and Eomes (53-4875-82, eBioscience) expression.
9
CD8+ Tem Cell Phenotyping by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We used flow cytometry to isolate and count CD8+ Tem cells. Cells were labeled with the following fluorescently conjugated monoclonal antibodies: anti-mouse CD45-PE (12045181, eBioscience, San Diego, CA, USA), anti-mouse CD8a-APC (17008181, eBioscience), anti-mouse CD44-PE (25044181, eBioscience), and anti-mouse CD62L (104432, BioLegend, San Diego, CA, USA). After the CD8+ Tem cells were sorted by flow cytometry, they were stained with antibodies against mTOR (ab87540, Abcam, Cambridge, MA, USA), interferon γ (IFN-γ) (11731181, eBioscience), T-bet (ab91109, Abcam), S6K (ab32529, Abcam) and Eomes (53-4875-82, eBioscience).
10
Isolation and Characterization of CD8+ Tem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells were isolated from blood samples and counted by flow cytometry. Cells were then labeled with the following fluorescence-conjugated monoclonal antibodies: anti-rat CD45 PE (12-0451-81, eBioscience, San Diego, CA, USA), anti-rat CD8a APC (17-0081-81, eBioscience), anti-rat CD44 PE (25-0441-81, eBioscience), and anti-rat CD62L (104432, Biolegend). CD8+ Tem were sorted by flow cytometry (EPICS-XL, Beckman-Coulter, Indianapolis, IN, USA) and then stained for interferon (IFN)-γ (11-7311-81, eBioscience), mTOR (ab87540, Abcam, Cambridge, MA, USA), and S6K (ab32529, Abcam) expression.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!