MG-63 and Saos-2 cells infected with LV-shCNN3 or LV-NC were seeded (4 × 105 cells/well) in 6-well plates. After being cultured for 48 h, cells were harvested, and the total protein content was isolated with RIPA buffer. After protein quantification, 30 μg of total protein was used for western blotting, which was conducted according to the methods described previously [33 (link)]. The primary antibodies used in this study were purchased from Abcam (Cambridge, MA, USA) and their details are as follows: anti-CNN3 (1:2000, ab151427), anti-MMP9 (1:1500, ab76003), anti-VEGF (1:5000, ab52917), anti-vimentin (1:1000, ab92547), anti-E-cadherin (1:1000, ab231303), p-p38 (1:1000, ab45381), p38 (1:1000, ab32142), ERK1/2 (1:1000, ab115799), p-ERK1/2 (1:800, ab214362), and anti-GAPDH (1:5000, ab181602).
Ab115799
Manufactured by Abcam
Sourced in United Kingdom
Ab115799 is a lab equipment product offered by Abcam. It serves as a tool for researchers in their scientific investigations, but a detailed description of its core function is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Ab214362, by Abcam (1 mentions)
Ab32142, by Abcam (1 mentions)
Ab76003, by Abcam (1 mentions)
Ab231303, by Abcam (1 mentions)
Ab45381, by Abcam (1 mentions)
Ab52917, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Ab92547, by Abcam (1 mentions)
Ab176564, by Abcam (1 mentions)
Ab52918, by Abcam (1 mentions)
Ab108319, by Abcam (1 mentions)
Ab201015, by Abcam (1 mentions)
Ab96379, by Abcam (1 mentions)
Ab32091, by Abcam (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Anti ng2 antibody, by Santa Cruz Biotechnology (1 mentions)
Ab50011, by Abcam (1 mentions)
Ab31392, by Abcam (1 mentions)
Anti c myc, by Cell Signaling Technology (1 mentions)
Anti α tubulin antibody, by Proteintech (1 mentions)
5 protocols using ab115799
1
Western Blot Analysis of CNN3 Regulation
2
Immunoblotting Analysis of Neurotropic Factors
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A total of 1 mL of RNA immunoprecipitation lysis buffer and 10 μL of phenylmethanesulfonyl fluoride were used to decompose and differentiate cells and tissues in each group. After the cells were sonicated and centrifuged, the supernatant was added with protein-loading buffer and treated at 100°C for 10 min. A total of 10 g of sample was loaded on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel for electrophoresis. After the electrophoretic separation, the protein was transferred to the PVDF membrane for 90 min. The sample was blocked with 5% skimmed milk powder for 60 min, and the primary antibody (NGF, ab52918; BDNF, ab108319; GDNF, ab176564; Spns2, ab82629; p-Erk1/2, ab201015; Erk1/2, ab115799; Abcam) was added to incubate for 2 h at room temperature. After washing, the horseradish peroxidase secondary antibody (ab6721) was added and placed at 4°C overnight. The membrane was washed three times by tris-buffered saline with Tween-20 and exposed in the ECL chemiluminescence kit (Pharmacia-Amersham, Piscataway, NJ, USA) darkroom. ImageJ software performed grayscale analysis. β-Actin was used as an internal reference to calculate the expression of each protein.
3
Protein Extraction and Western Blotting
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By reported procedures,18 (link) protein extraction and Western blotting were performed. The subfamily of pERK is ERK1 (pT202/pY204)+ Erk2 (pT185/pY187) (cat. no ab5001, dilution, 1:10,000; Abcam, Cambridge, MA, USA), the subfamily of ERK is ERK1+ ERK2 (cat. no ab115799, dilution 1:1,000; Abcam). The subfamily of pMEK is MEK1 (pS298) (cat. no ab96379, dilution 1:3,500; Abcam), and the subfamily of pMEK is MEK1 (cat. no ab32091, dilution 1:5,000; Abcam). β-Actin was considered as the internal control. All results were conducted in triplicate.
4
Antibody Immunodetection of Stem Cell Markers
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The anti-NG2 antibody (1:100; sc-166251) was from Santa Cruz Biotechnology (Heidelberg, Germany). The anti-α-tubulin antibody (1:1,000; 66031) was from Proteintech Germany GmbH (St. Leon-Rot, Germany). The antibodies anti-Akt1/2/3 (1:100; 4685), anti-pAkt (1:100; 4060) and anti-c-Myc (1:50; 5605) were from Cell Signaling (Frankfurt am Main, Germany). The anti-ERK1/2 antibody (1:100; ab115799), anti-pERK1/2 antibody (1:100; ab50011) and anti-PTEN antibody (1:50; ab31392) were from Abcam (Cambridge, UK). The peroxidase-labeled anti-rabbit antibody (1:1000; NIF 824) and peroxidase-labeled anti-mouse secondary antibody (1:1,000; NIF 825) were from GE Healthcare (Freiburg, Germany). The PE-labeled anti-chondroitin sulfate proteoglycan 4 antibody (1:30; NG2; 562415), PE-labeled anti-ITGB1 antibody (1:30; 556049), PE-labeled anti-PDGFRα antibody (1:30; 556002), and PE-labeled anti-PDGFRβ antibody (1:30; 55821) were from BD Biosciences (Heidelberg, Germany).
5
Western Blot Analysis of Brain Proteins
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After protein extraction from brain tissues, proteases and phosphatases were inhibited by the addition of a Complete Mini Protease Inhibitor Cocktail Tablet (Roche, Basel, Switzerland) and the phosphatase inhibitor phenylmethylsulfonyl fluoride (10 mM). The protein concentrations of the samples were assessed using the Bradford protein assay. Total proteins (50 µg) were heated at 90°C for 10 minutes and then separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were electrophoretically transferred onto nitrocellulose membranes, which were blocked using 5% skim milk. The membranes were then incubated with primary polyclonal antibodies recognizing EPOR (1:1000, FNab02816, Wuhan Fine Biotech, Wuhan, China), p-ERK1 (1:500, ab131438, Abcam, Cambridge, United Kingdom), ERK (1:1000, ab115799, Abcam), and CD34 (1:1000, AF-4117, R&D Systems, Minneapolis, MN) at 4°C overnight. Secondary antibodies (1:5000, ab6721 [Abcam] or 1:5000, sc-2352 [Santa Cruz, Dallas, TX]) were then reacted with the bound primary antibodies and visualized using enhanced chemiluminescence reagents (Pierce, Rockford, IL). The loading control comprised glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Image-J software was used for densitometry analysis.
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