Rabbit polyclonal anti-human MIIP antibody (HPA044948) was purchased from Sigma-Aldrich (St Louis, USA). The ITGB3 (#13166), ITGAV (#4711), β-catenin (#8480), FAK (#13009), p-FAK(Tyr 576/577, #3281), p-FAK(Tyr 397, #8556), AKT(#4691), p-AKT(Ser 473, #9271), E-cadherin (#3195), N-cadherin (#13116), Vimentin (#5741), Claudin-1(#13255), Myc-tag (#2278, #2276 S), Ubiquitin (#3933), and the HRP-conjugated goat anti-rabbit antibodies were obtained from Cell Signaling Technology (Beverly, USA). The VEGFA antibody (ab46154) was purchased from Abcam (Cambridge, UK). The HA antibody (#11867423001) was from Roche (Basel, Switzerland). The HRP-conjugated β-actin (HRP-66009) and GAPDH (HRP-60004) antibodies were obtained from Proteintech (Chicago, USA). The HRP-conjugated goat anti-mouse IgG were purchased from ZSGB-BIO (Beijing, China) respectively.
Gapdh hrp 60004
Manufactured by Proteintech
Sourced in United States
GAPDH (HRP-60004) is a primary antibody conjugated with Horseradish Peroxidase (HRP). It is commonly used as a loading control or reference protein in Western blotting experiments to normalize protein expression levels.
Automatically generated - may contain errors
Lab products found in correlation
N cadherin, by Cell Signaling Technology (3 mentions)
E cadherin, by Cell Signaling Technology (3 mentions)
Ubiquitin, by Cell Signaling Technology (2 mentions)
β catenin, by Cell Signaling Technology (2 mentions)
Vimentin, by Cell Signaling Technology (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Myc tag, by Cell Signaling Technology (1 mentions)
Itgb3, by Cell Signaling Technology (1 mentions)
Itgav, by Cell Signaling Technology (1 mentions)
P fak, by Cell Signaling Technology (1 mentions)
Claudin 1, by Cell Signaling Technology (1 mentions)
Hrp conjugated goat anti rabbit antibody, by Cell Signaling Technology (1 mentions)
Hrp conjugated goat anti mouse igg, by ZSGB-BIO (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
β catenin c2206, by Merck Group (1 mentions)
β actin hrp 60008, by Proteintech (1 mentions)
Protoporphyrin 9 zinc 2 znpp, by Merck Group (1 mentions)
Bibr1532, by Selleck Chemicals (1 mentions)
Ab39638, by Abcam (1 mentions)
Ab32360, by Abcam (1 mentions)
10 protocols using gapdh hrp 60004
1
Antibody Validation for Protein Analysis
2
Antibody selection for immunoblot analysis
3
Protein Expression Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extracts were resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membrane and blocked with 5% non-fat milk or bovine serum albumin. The desired bands were incubated with the appropriate primary and secondary antibodies and were subsequently visualized. The antibodies used in this study included anti-VEGFReceptor2 antibody (AB39638, Abcam, USA), the anti-NF-κB p50/105 antibody (AB32360, Abcam, USA), GAPDH HRP-60004 (Proteintech, USA), Anti-NF-κB p65 (phospho S536) antibody (ab76302, Abcam, USA), Anti-NF-κB p65 antibody (ab32536, Abcam, USA) and MAPK (Erk1/2) Antibody (8690, CST, USA), and Phospho-MAPK Rabbit mAb (2325, CST, USA).
4
Western Blot Analysis of Protein Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed with PBS and lysed in lysis buffer (20 mM Tris at pH 7.5, 150 mM NaCl, 1% Triton X-100, sodium pyrophosphate, β-glycerophosphate, EDTA, Na3VO4, leupeptin). The concentration of the protein was quantified using the BCA protein assay (Thermo Fisher Scientific, USA). Western blotting was implemented in accordance with the standard methods [47 (link)]. The antibodies used were as follows: PRMT5 (P0493, Sigma), GAPDH (HRP-60004, Proteintech), CASP1 (3866, CST), cleaved-CASP1 (4199, CST), H4R3me2s (ab5823, Abcam), cleaved-CASP3 (9661, CST), N-GSDMD (ab215203, Abcam), IL-1b (66737-1-Ig, Proteintech), and IL-18 (10663-1-AP, Proteintech). Quantifications of all western blotting signals with 3 repeated experiments are shown in Supplemental Fig. 2A–D as histograms.
5
Western Blot Analysis of Fibrosis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extracts were resolved by 8%∼15% SDS–polyacrylamide gel, transferred to PVDF membranes, and probed with antibodies against α-SMA (ab32575, Abcam; 1:2,000), collagenI(ab34710, Abcam; 1:5,000), collagen III (ab7778, Abcam; 1:5,000), SOCS1 (#3950, Cell Signaling Technology; 1:1,000), PPARγ (sc-7196, Santa Cruz; 1:200), phospho-STAT6 (sc-11762, Santa Cruz; 1:500), and STAT6 (#9362, Cell Signaling Technology; 1:1,000), HDAC1 (#5356, Cell Signaling Technology; 1:1,000), and HDAC2 (#5113, Cell Signaling Technology; 1:1,000), GAPDH(HRP-60004, Proteintech; 1:10,000). Peroxidase-conjugated secondary antibody (CST) was used as the secondary antibody and the antigen-antibody reaction was visualized by enhanced chemiluminescence assay (ECL, Thermo). Western blot quantification was performed with ImageJ software, analysing the intensity of the grey scale images. The images have been cropped for presentation. Full size images are presented in Supplementary Fig. 7 .
6
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For preparing proteins, cultured cells were lysed in radioimmune precipitation assay (RIPA) buffer plus phenylmethylsulfonyl fluoride (PMSF), protease and phosphatase inhibitors. Then the cell lysates were mixed with BCA solution and the protein concentration was determined by absorbance at 562 nm in SpectraMax M5 Multi-Mode Microplate Reader according to the instruction of the BCA protein assay kit (Beyotime, Haimen, China). Then 20 μg of protein extracts were subjected to western blot according to the standard procedure. The specific antibodies used in this study are as follows: β-Catenin (#8480), E-Cadherin (#3195), N-Cadherin (#13116), c-Myc (#9402), IgG (30000-0-AP), HA-Tag (#3724), FLAG (#14793), Ubiquitin (#3933), purchased from Cell Signaling Technology (Danvers, MA 01923, USA); c-Myc (10828-1-AP) and GAPDH (HRP-60004) purchased from Proteintech Group (Rosemont, IL 60018, USA). All antibodies were diluted to 1: 1000 in the blotting.
7
Antibody Validation for Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
AmotP130 antibody (sc‐166924) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); FAK (66258‐1‐lg) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH; HRP‐60004) were purchased from Proteintech (Wuhan, China); P‐FAK (ab81298) was purchased from (Abcam, Cambridge, MA, USA); vinculin, E‐cadherin, N‐cadherin, vimentin, RhoA, Rac1, Cdc42 and ROCK1 were purchased from Cell Signaling Technology (Danvers, MA, USA).
8
Quantitative Analysis of TPH2 in Piglet Brainstems
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Brainstems from euthanized piglets were prepared by homogenization in the presence of protease inhibitors (ThermoFisher Scientific, Waltham, MA, USA), and centrifuged to remove residual debris. The samples were heat-denatured at 95°C for 5 minutes with 5× SDS-PAGE loading buffer and fractionated on 10% SDS-PAGE gels (Bio-Rad, Hercules, CA, USA). GAPDH (HRP-60004, Proteintech, Manchester, UK) was used as a standard control protein. Following electrophoresis, proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk powder. Primary antibodies used Anti-TPH2 antibody (ab121013, Abcam, Cambridge, MA, USA), which use a concentration of 2
mg/mL.
mg/mL.
9
Investigating Sintilimab's Effects on TNBC Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MDA-MB-231 and 4T1 cells were purchased from Beijing Union Medical College Hospital Cell Resource Sharing Platform, SUM159 cells were a gift from Dr. Jianjun He. MDA-MB-231 was cultured in L-15 medium, SUM159 was cultured in DMEM/F-12 medium, and 4T1 was cultured in RPMI 1640 medium. All cell lines were stored in a thermostat incubator at 37 °C, 5 % CO2.
MDA-MB-231 and SUM159 were prepared extracting proteins after adding sintilimab at a concentration gradient of 0.5, 1, 2, and 4 μg/mL following with incubating for 24 h, and then, western blotting assay were conducted. ALDH1A1 antibody was purchased from Abcam (ab52492), PD-L1 antibody was purchased from Cell Signaling Technology (#13684), β-catenin (51067-2-AP) and GAPDH (HRP- 60004) antibodies were from Proteintech. Whole cell lysates were prepared with RIPA buffer, proteins were separated by SDS/PAGE gel, transferred to PVDF membrane (Millipore), incubated with primary antibody overnight, followed by HRP-conjugated secondary antibody (Proteintech, SA00001-2) and detected chemiluminescent signals.
MDA-MB-231 and SUM159 were prepared extracting proteins after adding sintilimab at a concentration gradient of 0.5, 1, 2, and 4 μg/mL following with incubating for 24 h, and then, western blotting assay were conducted. ALDH1A1 antibody was purchased from Abcam (ab52492), PD-L1 antibody was purchased from Cell Signaling Technology (#13684), β-catenin (51067-2-AP) and GAPDH (HRP- 60004) antibodies were from Proteintech. Whole cell lysates were prepared with RIPA buffer, proteins were separated by SDS/PAGE gel, transferred to PVDF membrane (Millipore), incubated with primary antibody overnight, followed by HRP-conjugated secondary antibody (Proteintech, SA00001-2) and detected chemiluminescent signals.
10
Comprehensive Antibody Reagents for Cellular Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies against p-STAT3 (Tyr705) (#9145), STAT3 (#9139), p-Smad2 (Ser465/467)/Smad3 (Ser423/425) (#8828), and Smad2/3 (#8685) were purchased from Cell Signaling Technology (Beverly, MA); antibodies against GDF11 (sc-81952) and SOCS3 (sc-51699) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies against Fbx32 (ab168372), CD31 (ab28364), and wheat germ agglutinin (ab178444) were purchased from Abcam (Cambridge, MA); recombinant GDF11 protein (1958-GD) were purchased from R&D Systems (Minneapolis, MN); antibodies against Trim63 (55456-1-AP), ubiquitin (10201-2-AP), iNOS (18985-1-AP), and GAPDH (HRP-60004) were purchased from ProteinTech group (Rosemont, IL); and antibodies against FoxO1 (BS1746) were purchased from bioworld (Bloomington, MN). Crotaline (C2401) were purchased from Sigma-Aldrich (St. Louis, MO). Stattic (S7024) were purchased from Selleck (TX, USA). Human GDF11 ELISA Kit (QC-GDF11-Hu), rat GDF11 ELISA Kit (QC-GDF11-Ra), and mouse GDF11 ELISA Kit (QC-GDF11-Mu) were purchased from QCHENG BIO (Shanghai, China). Human MSTN ELISA Kit (E-EL-H1437c) and human Activin A ELISA Kit (E-EL-H0003c) were purchased from Elabscience Biotechnology (Wuhan, China). MG-132 was purchased from Tocris Bioscience (Minneapolis, MN).
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!