Total proteins were extracted from HSCs with different treatments, equal amounts of soluble protein were separated via sodium dodecyl sulphate-polyacrylamide gel electrophoresis using 10% Tris-glycine mini-gels and transferred onto a nitrocellulose membrane (Bio-Rad).The primary antibodies were listed in Table S1 , GAPDH mAb (Santa Cruz, Biotechnology) was used as an internal control. Following incubation with horseradish peroxidase-conjugated secondary antibody (Zsbio, Beijing, China) for 2 h at room temperature, the bands were then tested by a chemiluminescent substrate ECL kit (Merck Millipore).
Gapdh mab
Manufactured by Santa Cruz Biotechnology
GAPDH mAb is a monoclonal antibody that specifically recognizes the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein. GAPDH is a key enzyme involved in the glycolytic pathway and is commonly used as a loading control in various biological assays.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase conjugated secondary antibody, by ZSGB-BIO (3 mentions)
Ecl kit, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Immobilon p transfer membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Imd 0354, by Selleck Chemicals (1 mentions)
Ecl reagent, by Cytiva (1 mentions)
Chemidoc system, by Bio-Rad (1 mentions)
Alexa fluor 488 nm, by Thermo Fisher Scientific (1 mentions)
Phalloidin tritc, by Merck Group (1 mentions)
6 protocols using gapdh mab
1
Western Blot Analysis of HSC Proteins
2
ETV2 Expression in hADSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was performed according to standard procedures elsewhere. The expression of ETV2 in ETV2-hADSCs was detected with anti-human ETV2 mAb (Abcam, Cambridge, MA). GAPDH mAb (Santa Cruz, Biotechnology) was used as an internal control. Following incubation with horseradish peroxidase-conjugated secondary antibody (Zsbio, Beijing, China) at room temperature for 2 h, the bands were detected by a chemiluminescent substrate ECL kit (Merck Millipore).
3
Western Blot Protein Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted from the treated cells or tissues, as indicated, using RIPA lysis buffer (Beyotime, Nanjing, China) containing a 1% protease inhibitor cocktail (Merck Millipore, Birrika, USA), and prepared for SDS-PAGE loading buffer (Abclonal, Wuhan, China). Proteins were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis using 10% Tris–glycine mini-gels and transferred onto a PVDF membrane (0.45 μm, Immobilon-P Transfer Membranes, Merck Millipore). The primary antibodies used are listed in Additional file 1 : Table S2, and the corresponding dilutions are 1:1000 according to the antibody instructions. GAPDH mAb (Santa Cruz, Biotechnology) was used as the loading control for all experiments. Following incubation with horseradish peroxidase-conjugated secondary antibody (Zsbio, Beijing, China) for 2 h at room temperature, the bands were then tested by a chemiluminescent substrate ECL kit (Yamei, Shanghai, China).
4
Immunoblotting Assay for NF-κB Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti-phospho-NF-κB p65 (Ser536) rabbit polyclonal antibody (pAb) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-NF-κB p65 rabbit pAb was obtained from Abcam (Cambridge, MA, USA). IκBα monoclonal antibody (mAb), His mAb, and Flag mAb were purchased from Cell Signaling Technology (Danvers, MA, USA). GAPDH mAb, tubulin mAb, VEGF mAb, smooth muscle actin (SMA) pAb, horseradish-peroxidase-labeled goat anti-mouse and goat anti-rabbit IgG were purchased from Santa Cruz Biotechnology (Santa Cruz). Western blotting was carried out as described previously (46 (link),47 (link)). IMD 0354, an IKKβ inhibitor, was obtained from Selleck Chemicals (Shanghai, China).
5
Western Blot Analysis of C-myc Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were prepared by lysing cells in RIPA buffer (10 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% Na deoxycholate), supplemented with protease inhibitor cocktail (Sigma). Protein concentration of whole cell lysates was determined by the BSA assay using the BSA kit (Pierce), and then equal protein amounts were heated to 95 °C for 5 min with sodium dodecyl sulfate (SDS) sample buffer (25 ml glycerol, 31.2 ml Tris buffer, 7.5 ml SDS, a dash of bromophenol blue/100 ml) and run on 15% SDS polyacrylamide gel electrophoresis (SDS-PAGE). Protein samples were then blotted onto PVDF membranes (Immobilon P, Watford, United Kingdom). The membranes were incubated in blocking solution (5% non-fat milk in PBS) for 1 h and then in primary antibody (anti-human C-myc pAb (Abcam; at dilution of 2:1,000) or GAPDH mAb (Santa Cruz; at dilution of 20:1,000)) overnight. After 3 × 10 min washes in TBS (0.1% Tween-20 in PBS), the membrane was incubated for 1 h at room temperature with horseradish-peroxidase–linked (HRP) secondary anti-rabbit or anti-mouse Ab (1:1,000 dilution in PBS). Signals on the membrane were developed using ECL reagent (Amersham, CA, United States) and then were imaged with the Chemidoc system (Bio-Rad, United States).
6
Immunofluorescence Analysis of Mitochondrial Networks
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence assays were performed on PBMC cultured on glass coverslips with pro-osteoclastogenic medium or control medium. Cells were fixed in 2% paraformaldehyde. Permeabilization was performed with 0.5% Triton X-100 in HEPES and blocked with 1% BSA. We used anti-mitochondria MAb against the surface of intact mitochondria (clone 113-1, MAB1273, Merck Millipore, 1:50), anti-Pyruvate kinase isozymes M2 (PKM2) polyclonal antibody (Cell Signaling, 1:200) ; and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) MAb (Santa Cruz, 1:50). As secondary antibodies we used anti-mouse or anti-rabbit antibodies Alexa Fluor 488 nm (Life Technologies). For the staining of polymeric actin (F-actin) we used phalloidin-TRITC (Sigma, 0.5 μg/mL).
Nuclei were stained with Hoechst 33258. Mitochondrial mass index was quantified as the ratio of the area with a fluorescent signal to the total area of the single cell by using NIS Element image software BR4.00.00 (Nikon). Cells were counted in 28 different random fields. Mitochondria networks were observed by confocal microscope (Nikon TI-E).
Nuclei were stained with Hoechst 33258. Mitochondrial mass index was quantified as the ratio of the area with a fluorescent signal to the total area of the single cell by using NIS Element image software BR4.00.00 (Nikon). Cells were counted in 28 different random fields. Mitochondria networks were observed by confocal microscope (Nikon TI-E).
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!