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).
Ab32028
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab32028 is a laboratory equipment product manufactured by Abcam. It is designed for a core function related to biological research applications. No further details are available for this product at this time.
Automatically generated - may contain errors
Lab products found in correlation
Ab109268, by Abcam (41 mentions)
Ab8805, by Abcam (11 mentions)
Ab38449, by Abcam (11 mentions)
Ab81283, by Abcam (7 mentions)
Ab192890, by Abcam (7 mentions)
Ab179463, by Abcam (7 mentions)
Pvdf membrane, by Merck Group (7 mentions)
Ab191606, by Abcam (6 mentions)
Ab32529, by Abcam (5 mentions)
Ab32503, by Abcam (5 mentions)
Ab205718, by Abcam (5 mentions)
Ab59208, by Abcam (5 mentions)
Ab209847, by Abcam (4 mentions)
Ab181602, by Abcam (4 mentions)
Ab137133, by Abcam (4 mentions)
Ab133448, by Abcam (4 mentions)
Ripa buffer, by Beyotime (4 mentions)
Ab32359, by Abcam (4 mentions)
Ab8227, by Abcam (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
71 protocols using ab32028
1
Quantifying Protein Expression in Immune Cells
2
Immunohistochemical Detection of TLR4, NF-kB, and mTOR
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The main steps of immunohistochemical detection included dewaxing, hydration, 3% hydrogen peroxide treatment, high temperature antigen retrieval, goat serum sealing, primary antibody 4°C overnight, rewarming PBS wash, secondary antibody drop, DAB color development, hematoxylin staining, hydrochloric acid and alcohol differentiation, tap water wash, dehydration, and sealing. Images were captured under a microscope, and the Image-Pro Plus 6.0, Media Cybernetics system was used to detect absorbance and determine integrated option density (IOD)/area of TLR4 (ab22048, Abcam, Cambridge, MA, USA), NF-κB p65 (ab16502, Abcam), or mTOR (ab32028, Abcam), and to perform statistical processing.
3
Quantification of mTOR Signaling Pathway
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The cells were rinsed twice using PBS, harvested, pelleted by centrifugation, and lysed in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl at PH 7.4), combined with a protease inhibitor cocktail and phosphatase inhibitors. Soluble proteins (20–30 μg) were subjected to SDS-PAGE, then transferred to a PVDF membrane, blocked with 5% non-fat milk in TBS-with 0.05% Tween-20 for 1 h, and incubated overnight with primary antibodies, including phosphorylated eukaryotic translation initiation factor 4E binding protein 1 (p-4EBP1) (bs-14550R, Bioss, Beijing, China), 4EBP1 (60246-1-Ig, Proteintech, USA), p-Seventy kilodalton ribosomal protein S6 kinase 1 (P70S6K1) (ab2571, Abcam, UK), P70S6K1 (14485-1-AP, Proteintech, USA), P-mTOR (ab109268, Abcam, UK), and mTOR (ab32028, Abcam, UK), followed by horseradish peroxidase-linked secondary antibodies. The protein bands were visualized using a chemiluminescent reagent (Pierce, Rockford, IL) with a digital luminescent image analyzer LAS-1000 (Fujifilm, Japan). The resultant signals were quantified using Alpha Imager 2200 software (Alpha Innotech Corporation, San Leandro, CA) and normalized against the internal control, GAPDH (10494-1-AP, Proteintech, USA).
4
Western Blot Analysis of Signaling Proteins
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Cells were washed in phosphate-buffered saline (PBS) and lysed using radioimmunoprecipitation assay buffer (Invitrogen, Carlsbad, CA) supplemented with a protease inhibitor cocktail (Roche, Pleasanton, CA, USA). The protein concentration was evaluated using a bicinchoninic acid protein assay kit (Bioswamp, PAB180007, Wuhan, China). Equivalent amounts of proteins (30 μg) from each sample were subjected to sodium dodecyl-polyacrylamide electrophoresis, transferred to a polyvinylidene fluoride membrane, blocked in 5% fat-free milk for 2 hours at room temperature, and incubated with the following primary antibodies: PTEN (ab32199, 1:10,000, abcam), PI3K (ab191606, 1:10,000, abcam), p-PI3K (ab182651, 1:10,000, abcam), mTOR (ab32028, 1:10,000, abcam), p-mTOR (ab109268, 1:10,000, abcam), GLUT1 (ab652, 1:10,000, abcam), LDHB (ab85319, 1:10,000, abcam), HK2 (ab209847, 1:10,000, abcam), PKM2 (ab150377, 1:10,000, abcam), or GAPDH (PAB36264, 1:10,000, Bioswamp). Then, the membranes were washed with Tris-buffered saline and incubated with goat anti-rabbit IgG secondary antibody (SAB43711, 1:10,000, Bioswamp) for 2 h at room temperature. An enhanced chemiluminescence kit (Pierce) was used to detect specific bands and autoradiograms were quantified by densitometry (Quantity One software, Bio-Rad, Hercules, CA, USA) using GAPDH as a control. For each group, the quantification was triplicated.
5
Autophagic Protein Expression Analysis
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Proteins were extracted from the brain samples using a Proteins Extraction Kit (CWBIO, China) and quantified with BCA-Reagents (CWBIO, China). Proteins were separated by SDS–PAGE at 160 V on a 12% gel (CWBIO, China) for 1 h and then transferred to a 0.22 μm nitrocellulose filter membrane at 200 mA for 3 h. The membranes were incubated with autophagy related primary antibodies diluted as follows: anti-CST3 antibody (ab24327; Abcam, UK), anti-mTOR antibody (ab32028; Abcam, UK), anti-mTOR (phospho S2448) antibody (ab109268; Abcam, UK), anti-LC3 antibody (ab192890; Abcam, UK), anti-Beclin-1 antibody (3495, CST, USA) diluted 1:1,000, and anti-GAPDH antibody (ab181602; Abcam, UK) diluted 1:2,000. Membranes were then incubated with secondary antibodies diluted at 1:5,000. The membranes were washed and bands were visualized with enhanced chemiluminescence immunoblotting detection reagents (Thermo Fisher Scientific, USA). For autophagic protein, bafilomycin A1 was used at a final concentration of 200 nM and added during the last 3 h. Images were gotten by Image Lab Software (BIO-RAD, USA), and semiquantitative analysis was performed by normalizing the protein bands to the internal control GAPDH.
6
Western Blot Analysis of mTOR Pathway
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Three pairs ccRCC and adjacent tissues were chosen for WB analysis (Yuan et al., 2020 (link)). Membranes were incubated with the desired primary antibodies: mTOR (1:2,000, rabbit, ab32028; Abcam, Cambridge, MA, USA), p-mTOR (Ser2448, 1:2,000, rabbit, ab109268; Abcam, Cambridge, MA, USA), anti-Actin (1:1,000, mouse, BM0627; BOSTER, Beijing, China), the corresponding secondary antibodies (SA00001-2 & SA00001-1; Proteintech, Wuhan, China) were diluted to 1:5,000, and enhanced chemiluminescence (Amersham Imager 600; Marlborough, MA, USA) was used for immunodetection as previously described (Yuan et al., 2020 (link)).
7
Immunoblotting Analysis of Signaling Proteins
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The treated cells were washed twice with cold 1X phosphate-buffered saline, lysed with RIPA lysate buffer for 20 min, and centrifuged (12,000 rpm, 10 min, 4°C) to obtain the supernatant. The loading buffer was added, and the supernatant was denatured at 95°C for 5 min until protein denaturation was achieved. Electrophoresis analysis of protein samples (10 μL/well) was performed using 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Proteins were transferred onto nitrocellulose membranes (Millipore, Burlington, MA, USA). The blots were blocked with 5% nonfat milk powder for 1 h and incubated with corresponding primary antibody at 4°C overnight. On the second day, the blots were cleaned with 1X tris-buffered saline and incubated with the appropriate secondary antibodies at room temperature for 1 h. Protein brands were detected by chemiluminescence (Millipore, Burlington, USA), and the expression level was determined using Image J.
Primary antibodies against JAK (ab133666), anti-p-JAK (ab138005), anti-STAT1 (ab109320), anti-p-STAT1 (ab109461), mTOR (ab32028), p-mTOR (ab109268), p-AKT (ab105731), Bax (ab32503), and Bcl2 (ab182858) were purchased from Abcam (Cambridge, UK). The following secondary antibodies were purchased from GenScript (Piscataway, NJ, USA): goat anti-rabbit IgG (H&L) and goat anti-mouse IgG (H&L).
Proteins were transferred onto nitrocellulose membranes (Millipore, Burlington, MA, USA). The blots were blocked with 5% nonfat milk powder for 1 h and incubated with corresponding primary antibody at 4°C overnight. On the second day, the blots were cleaned with 1X tris-buffered saline and incubated with the appropriate secondary antibodies at room temperature for 1 h. Protein brands were detected by chemiluminescence (Millipore, Burlington, USA), and the expression level was determined using Image J.
Primary antibodies against JAK (ab133666), anti-p-JAK (ab138005), anti-STAT1 (ab109320), anti-p-STAT1 (ab109461), mTOR (ab32028), p-mTOR (ab109268), p-AKT (ab105731), Bax (ab32503), and Bcl2 (ab182858) were purchased from Abcam (Cambridge, UK). The following secondary antibodies were purchased from GenScript (Piscataway, NJ, USA): goat anti-rabbit IgG (H&L) and goat anti-mouse IgG (H&L).
8
Protein Expression Analysis in NSCLC
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To prepare the protein samples for loading, transfected H1975 and A549 cells or tumor tissues were lysed using a radio-immunoprecipitation assay. After quantification, 40 µg of protein samples were subjected to separation through sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). After blocking in non-fat milk, the membranes were incubated in Tris Buffered Saline Tween (TBST) containing primary antibody against Ki-67 (ab92742; Abcam), matrix metalloproteinase 9 (MMP-9; ab38898; Abcam), HK2 (ab209847; Abcam), phosphorylated protein kinase B (p-AKT; ab192623; Abcam), AKT (ab179463; Abcam), p-mammalian target of rapamycin (p-mTOR; ab109268; Abcam), mTOR (ab32028; Abcam), or GAPDH (ab181602; Abcam) and then incubated with secondary antibody (ab205718; Abcam). The blots were visualized using a chemiluminescence kit (Merck Millipore, Darmstadt, Germany). GAPDH served as a control.
9
Protein Analysis of Cellular Pathways
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High-glucose Dulbecco's modified Eagle's medium (H-DMEM) was obtained from Basalmedia Technologies (Shanghai, China) and fetal bovine serum (FBS) was obtained from Bailing Biotechnology (Lanzhou, China). TurboFect™ transfection reagent (R0531) and Pierce™ Protein G Magnetic Beads were from Thermo Fisher Scientific (Waltham, NY, USA). Cholesterol was obtained from Sigma-Aldrich (St. Louis, MO, USA). The primary antibodies, rabbit monoclonal anti-AKT1 (ab81283), anti-AKT-1/2/3 (ab179463), anti-mTOR/p-mTOR (ab32028, ab109268), anti-HO-1 (ab52947), anti-Aconitase2 (Aco2) (ab129069), anti-lactate dehydrogenase (LDH) (ab134187), anti-pyruvate dehydrogenase E-1β (PDHE-1β) (ab155996), anti-nuclear factor erythroid 2-like 2 (Nrf2) (ab62352) and anti-EBP (ab135745) were purchased from Abcam (Cambridge, MA, USA). Anti-histone H3.1 was obtained from Abmart. Anti-malic dehydrogenase (MDH2) (#8610) was obtained from Cell Signaling Technology. The anti-rabbit HRP-labeling and the anti-rabbit DyLight™ 488- and anti-mouse DyLight™ 633-labeled secondary antibodies were from KPL.
10
Western Blot Analysis of Spinal Cord Proteins
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Spinal cord tissue and cells were lysed by RIPA with phosphatase inhibitors and protease inhibitors cocktail, and protein concentration was determined using a bicinchoninic acid reagent (ThermoFisher). Equal amount of proteins was separated using 8–12% SDS-PAGE gels, and transferred to polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA). Then the membranes were blocked with 5% nonfat milk and incubated with primary antibodies: Mettl14 (1:1000, #51,104, CST, Beverly, MA, USA), MAP2 (1 µg/mL, ab11267, Abcam), acetyl-α-tubulin (0.06 µg/mL, ab24610, Abcam), Iba-1 (1:500, ab178846, Abcam), RASD1 (0.4 µg/mL, NBP1-91830, Shanghai Univ Biotechnology Co., Ltd., Shanghai, China), p-4EBP1 (1:1000, #2855, CST), t-4EBP1 (1:5000, ab32024, Abcam), p-mTOR (1:1000, ab109268, Abcam), t-mTOR (1:1000, ab32028, Abcam), and secondary antibodies (1:2000, ab205718 or 1:2000, ab205719; Abcam). The bands were examined by the ChemiDic XRS + Imaging System (Bio-Rad), and band intensities were analyzed with Image Lab 3.0 software (Bio-Rad). Experiments were performed at least three times.
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