As described by Feng et al [33 (link)]., cells were collected and immersed in RIPA lysis buffer (Solarbio, Beijing, China) and supplemented with phenylmethanesulfonyl fluoride. A BCA Protein Assay Kit was used to determine the protein concentration. Equal amounts of protein were subjected to 10% SDS-PAGE and then transferred to a polyacrylamide difluoride membrane. After blocking with 5% nonfat milk for 2 h and subsequent incubation with primary antibodies at 4°C overnight, membranes were probed with HRP-labeled secondary antibody (ab150077; Abcam) at room temperature for 2 h. The immunoreactive bands were detected with an enhanced chemiluminescence (ECL) system (Pierce). The following primary antibodies were used in this study: anti-IRS1 (ab40777; Abcam) and anti-GAPDH (ab181602; Abcam).
Ab40777
Manufactured by Abcam
Sourced in United States
Ab40777 is a primary antibody that recognizes amyloid beta (Aβ) peptide. This antibody is designed for use in various research applications, including immunohistochemistry, Western blotting, and ELISA.
Automatically generated - may contain errors
Lab products found in correlation
Labvision autostainer 480s, by Thermo Fisher Scientific (2 mentions)
Ab39398, by Abcam (2 mentions)
Immuno logic c dpvb blocking and polymer poly hrp gam r r igg, by Immunologic (2 mentions)
Ab9485, by Abcam (2 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Ab181602, by Abcam (1 mentions)
Ab150077, by Abcam (1 mentions)
Ab134175, by Abcam (1 mentions)
Ab40772, by Abcam (1 mentions)
Ab76151, by Abcam (1 mentions)
Ab76011, by Abcam (1 mentions)
Ab196575, by Abcam (1 mentions)
Ab137528, by Abcam (1 mentions)
Ab92547, by Abcam (1 mentions)
Ab32572, by Abcam (1 mentions)
Ab32072, by Abcam (1 mentions)
D110016, by Sangon (1 mentions)
Ab191606, by Abcam (1 mentions)
Imagej software, by IBM (1 mentions)
Ab38449, by Abcam (1 mentions)
8 protocols using ab40777
1
Western Blot Analysis of IRS1 and GAPDH
2
Immunohistochemical Analysis of pIGF-1R and IRS-1
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Tumors were pathologically identified and classified according to WHO guidelines. Briefly, 3 μm thick sections of FFPE tumors were deparaffinized. For pIGF-1R staining, antigen retrieval was performed by boiling the section in citrate buffer at pH 6 for 25 minutes. For IRS-1 staining, no pretreatment was necessary. Primary antibodies were used as follows: pIGF-1R (pY1161) (Ab39398, 1:100, pH 6, Abcam Inc.), IRS-1 (Ab40777, 1:100, Abcam Inc.). Corresponding secondary antibodies and detection kits were used (Enhancer: post antibody blocking for Bright Vision plus; Immuno Logic c-DPVB blocking and Polymer: Poly-HRP-GAM/R/R IgG; Immuno Logic c-DPVB999HRP, BrightVision+ cat #DPVB999HRP, ImmunoLogic, Duiven, The Netherlands, www.immunologic.nl ) and stained on an automated stainer (LabVision Autostainer 480S, Thermo Scientific). Staining intensities were individually evaluated by 3 independent observers.
3
Western Blot Analysis of Signaling Proteins
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Western blotting was performed using the primary antibodies, anti‐SH2B1 (1:1000, ab196575, Abcam, Cambridge, MA), anti‐E‐cadherin (1:3000, ab40772, Abcam), anti‐N‐cadherin (1:1000, ab76011, Abcam), anti‐Vimentin (1:2000, ab92547,Abcam), anti‐β‐catenin (1:5000, ab32572, Abcam), anti‐TCF‐4 (1:25000, ab76151,Abcam), anti‐CyclinD1 (1:10000, ab134175, Abcam), anti‐C‐Myc (1:1000, ab32072, Abcam), anti‐IRS1 (1:1000, ab40777), anti‐DVL‐2 (1:500, ab137528), antibodies (Abcam), and anti‐GAPDH antibody (1:3000. D110016, Sangon Biotech, Shanghai, China). Following the Western blotting assay, the membranes were stripped, and the band intensities were relative to GAPDH. The quantification of protein bands was performed using ImageJ software.
4
Western Blot Analysis of Insulin Signaling Proteins
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Radioimmunoprecipitation assay buffer (RIPA) (Sangon, China) was used to extract
proteins from cultured cells and the concentration was determined using a
bicinchoninic acid kit (Sangon Biotech Co., Ltd.). Total protein (10 µg) was
resolved by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS-PAGE). The proteins were transferred to polyvinylidene difluoride (PVDF)
membranes. Non-fat milk (10%) in PBS buffer (90%) was used to block the
membranes at room temperature for 1 h. Then, the membranes were incubated with
primary antibody at 4°C overnight and secondary antibody at room temperature for
2 h. The immunoreactive bands were developed using an enhanced chemiluminescence
kit (Santa Cruz, USA, sc-2048) and photographed on a gel imager system (Bio-Rad,
USA). The densitometry analysis was performed with ImageJ software (IBM, USA).
The antibodies used in this study were as follows (all from Abcam, USA): GAPDH
(1:2500; ab9485; USA); C/EBP-α (1:1000; ab40761); PPAR-γ (1:1000; ab178860);
C/EBP-α (1:1000; ab40761;); IRS (1:10000; ab40777); p-IRS (1:10000; ab109543);
AKT (1:500; ab8805); p-AKT (1:500; ab38449); FoxO1 (1:1000; ab179450); p-FoxO1
(1:1000; ab259337); PI3K (1:1000; ab191606); p-PI3K (1:1000; ab278545); β-actin
(1:1000; ab8226). In addition, HRP-linked secondary antibody was used (1:3000;
Cell Signaling #7074, USA).
proteins from cultured cells and the concentration was determined using a
bicinchoninic acid kit (Sangon Biotech Co., Ltd.). Total protein (10 µg) was
resolved by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS-PAGE). The proteins were transferred to polyvinylidene difluoride (PVDF)
membranes. Non-fat milk (10%) in PBS buffer (90%) was used to block the
membranes at room temperature for 1 h. Then, the membranes were incubated with
primary antibody at 4°C overnight and secondary antibody at room temperature for
2 h. The immunoreactive bands were developed using an enhanced chemiluminescence
kit (Santa Cruz, USA, sc-2048) and photographed on a gel imager system (Bio-Rad,
USA). The densitometry analysis was performed with ImageJ software (IBM, USA).
The antibodies used in this study were as follows (all from Abcam, USA): GAPDH
(1:2500; ab9485; USA); C/EBP-α (1:1000; ab40761); PPAR-γ (1:1000; ab178860);
C/EBP-α (1:1000; ab40761;); IRS (1:10000; ab40777); p-IRS (1:10000; ab109543);
AKT (1:500; ab8805); p-AKT (1:500; ab38449); FoxO1 (1:1000; ab179450); p-FoxO1
(1:1000; ab259337); PI3K (1:1000; ab191606); p-PI3K (1:1000; ab278545); β-actin
(1:1000; ab8226). In addition, HRP-linked secondary antibody was used (1:3000;
Cell Signaling #7074, USA).
5
Immunohistochemistry and Cell Signaling Assays
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The IRS-1 antibody for immunohistochemistry analysis was obtained from Abcam (#ab40777; Abcam, Boston, MA, USA). Similarly, the caspase 3 antibody was obtained from Abcam (#ab184787). Cell signaling (Danvers, MA, USA) provided antibodies for caspase 9 (#9502S); hexokinase (#2867S), STAT3 (#4904S), phospho STAT 3, (#9131S), cleaved PARP (#5625S), IRS-1 for western blotting (#2382S), phospho p38 MAP Kinase (#9215S), p38 MAP Kinase (#9212S), phospho AKT (#3787S), AKT (#9272S), phospho Erk1/2(#4376S), Erk1/2 (#9102S) and IGF-1R (#3027S). Phospho IGF-1R antibody was obtained from Sigma Aldrich (SAB4300652; Millipore Sigma, Burlington, MA, USA). The Actin antibody was obtained from Santa Cruz Biotechnology (#sc-47778; Santa Cruz, Dallas, TX, USA). NT157 was obtained from Selleckchem (#NM-4126; Houston, TX, USA).
Cell migration assay plates were obtained from Corning (#354578; Corning, Glendale, AZ, USA) and the staining kit was obtained from Siemens (#B4132-1A; Siemens Health care Diagnostic Inc., Newark, DE, USA). Methylthiazole tetrazolium (MTT) reagent was obtained from EMD Millipore Corp (#475989; United Kingdom). Propidium iodide was procured from Sigma Aldrich (#81845). Ribonuclease A (RNase) was obtained from Sigma-Aldrich (#R4642).
Cell migration assay plates were obtained from Corning (#354578; Corning, Glendale, AZ, USA) and the staining kit was obtained from Siemens (#B4132-1A; Siemens Health care Diagnostic Inc., Newark, DE, USA). Methylthiazole tetrazolium (MTT) reagent was obtained from EMD Millipore Corp (#475989; United Kingdom). Propidium iodide was procured from Sigma Aldrich (#81845). Ribonuclease A (RNase) was obtained from Sigma-Aldrich (#R4642).
6
Western Blot Analysis of Metabolic Proteins
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Total protein was extracted from cells and liver tissues using RIPA buffer. Protein concentrations were determined with a Pierce BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL). Next, 20 ug protein was loaded and separated with 12% SDS gels and transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA). Following transfer, membranes were blocked with 10% skimmed milk and incubated with the appropriate 1:1000 primary antibodies against Sirt1 (ab189494, Abcam), FGF-21 (ab171941, Abcam), PTP1B (ab244207, Abcam), p-PKB (ab175349, Abcam), p-mTOR2 (ab134903, Abcam), IRS1 (ab40777, Abcam), GLUT4 (ab654, Abcam) and GAPDH (ab9485, Abcam) at 4°C overnight. Furthermore, the corresponding HRP-conjugated secondary antibodies were used to detect the protein expression. Chemiluminescent film (Roche) was applied for assessment of protein expression with ImageJ software (NIH).
7
Immunohistochemical Analysis of pIGF-1R and IRS-1
8
Quantifying Liver and Muscle Protein Profiles
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Protein samples were extracted from the liver and muscle tissues were quantified using the BCA Protein Concentration Assay Kit (Kanka Century) and boiled to denature the proteins. Proteins isolated and purified from the 10% Bio‐Rad protein gel were transferred to a PVDF membrane using a semidry instrument (Bio‐Rad) and blocked with 5% skimmed milk for 2 hours. The target protein bands were cut according to the size of the marker, incubated at 4°C overnight with the corresponding primary antibodies IRS1 (abcam, ab40777, 1:1000), IRS2 (abcam, ab46811, 1:1000), PI3 kinase p85α (abcam, ab40755, 1:500), and GSK3β (abcam, ab131356, 1:1000), and later washed three times with TBST. Control samples were incubated with anti‐β‐actin (GeneTex, GTX629630, 1:4000) for 1 hour at room temperature and washed three times with TBST. Images were successfully collected, and the data were analyzed using the ECL chemiluminescence method and the Bio‐Rad ChemiDoc Touch system.
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