Total protein from the transfected MC3T3-E1 cells was extracted using RIPA lysis buffer (Millipore) and the concentration was assessed using a bicinchoninic acid (BCA) kit (BCA1-1KT, Sigma, St Louis, MO, USA). Protein samples (30 μg) were separated by 10% SDS-PAGE, and subsequently blotted onto a PVDF membrane (EMD Millipore, Billerica, MA, USA). After incubation at 4°C overnight with specific primary antibodies, the blot was probed with corresponding horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h. Then, the immunoreactive bands were visualized with the ECL detection reagent (Millipore). GAPDH was used as the internal loading control. The antibodies used were: ALP (1: 1000, Cell Signaling Technology, Inc.), OCN (1: 1000, Cell Signaling Technology, Inc.), Runx2 (1: 1000, Cell Signaling Technology, Inc.), Collagen1 (1: 1000, Cell Signaling Technology, Inc.), BMP2 (1: 1000, Millipore), BMP4 (1: 1000, Millipore), BMP6 (1: 1000, Millipore), BMP7 (1: 1000, Millipore), mouse anti-rabbit secondary antibody (1: 10 000, Santa Cruz, sc-2357), and rabbit anti-mouse secondary antibody (1: 10 000, Santa Cruz, sc-358914).
Rabbit anti mouse secondary antibody
Manufactured by Santa Cruz Biotechnology
Sourced in China, United States, Germany
The Rabbit anti-mouse secondary antibody is a reagent used in various immunoassay techniques. It is a polyclonal antibody produced in rabbits that binds to mouse primary antibodies, allowing for their detection and visualization in experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (1 mentions)
Ecl detection reagent, by Merck Group (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Collagen 1, by Cell Signaling Technology (1 mentions)
Bone morphogenetic protein 4 (bmp4), by Merck Group (1 mentions)
Runx2, by Cell Signaling Technology (1 mentions)
Donkey anti goat secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Mouse anti map lc3 antibody sc 376404, by Santa Cruz Biotechnology (1 mentions)
Bx 795 s1274, by Selleck Chemicals (1 mentions)
Acetylated lysine antibody 9441, by Cell Signaling Technology (1 mentions)
Mouse interferon β elisa kit, by Cusabio (1 mentions)
Goat anti rabbit secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ecl chromogenic substrate, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Avantor (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Glutathione (gsh), by Abcam (1 mentions)
Superoxide dismutase, by Abcam (1 mentions)
Catalase, by Abcam (1 mentions)
9 protocols using rabbit anti mouse secondary antibody
1
Western Blot Analysis of Osteogenic Markers
2
Antibody Characterization for Innate Immunity
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The mouse anti-Map-LC3 antibody (sc-376404), the rabbit polyclonal anti-mouse AIM2 antibody (sc-137967) and the goat polyclonal antibody TMEM173 (M-12) (sc-241049) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The rabbit anti-mouse β-actin (AP0060) was obtained from Bioworld Technology (Nanjing, Jiangsu, China). The rabbit Anti-NAK/TBK-1 antibody (ab40676) and rabbit anti-NAK/TBK-1 (phosphor S172) antibody (ab109272) were obtained from Abcam (Cambridge, UK). The rabbit TMEM173 polyclonal antibody (19851-1-AP) and rabbit IRF3 polyclonal antibody (11312-1-AP) are from Protein Tech (Wuhan, Hubei, China). The phosphor-IRF3 (Ser396) (4D4G) and the Acetylated-Lysine Antibody (9441) were from Cell Signaling Technology (Boston, Mass, USA). The goat anti-rabbit secondary antibody, rabbit anti-mouse secondary antibody and donkey anti-goat secondary antibody were obtained from Santa Cruz Biotechnology, Beijing ZSGB Biotechnology and Beijing Cowin Biotechnology (Beijing, China), respectively. The BX-795 (S1274) was from Selleckchem. The Fast Protein Precipitation and Concentration Kit were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Reagents and apparatus used in immunoblotting assays were obtained from Bio-Rad (Hercules, CA, USA). The mouse interferon-β ELISA kit (CSB-E04945m) was from Cusabio (Wuhan, Hubei, China).
3
FASN Protein Expression Analysis
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Proteins were extracted from cells using RIPA lysis buffer (Beyotime Institute of Biotechnology) containing PMSF (Amresco, LLC) at a dilution of 1:100. Protein concentrations were then measured via bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). Proteins were next resuspended in sample loading buffer, boiled for 5 min, and electrophoresed on a polyacrylamide 10% gel. The proteins were then transferred to a nitrocellulose membrane, which was blocked with 5% low-fat dried milk at room temperature for 1 h. The blots were then probed with specific antibody against FASN (1:1,000; cat. no. sc-48357; Santa Cruz Biotechnology, Inc.) overnight at 4°C, followed by rabbit-anti-mouse secondary antibody (1:2,000; cat. no. sc-358914; Santa Cruz Biotechnology, Inc.) at room temperature for 2 h. ECL chromogenic substrate (Beyotime Institute of Biotechnology) was then used for chemiluminescence imaging to measure protein levels.
4
Western Blot Analysis of MUC1 Expression
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Cellular lysate preparation and Western blotting was done as previously described [36 (link)]. A 5% SDS-PAGE was used and 15μgs of cell lysate was loaded. 1:10,000 TAB 004 mouse monoclonal anti-human MUC1 antibody was used to probe for MUC1-TR (18). TAB 004 recognizes the STAPPVHNV epitope within MUC1 TR [37 (link)]. Membranes were also probed for β-actin (Santa Cruz) to account for equal loading of the protein. Rabbit anti-mouse secondary antibody was used from Santa Cruz, at 1:5000. The NIH Imaging program was used to conduct densitometry analyses of immunoblots as shown in Supplementary Figure 1b . Results are presented as mean values of arbitrary densitometry units corrected for background intensity and normalized to the expression of b-actin.
5
Evaluating Apelin-13's Antioxidant Effects
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Apelin-13 was purchased from Sigma Aldrich (St Louis, MO). Malonaldehyde (MDA), catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), and enzyme-linked immunosorbent assay (ELISA) kits were bought from Abcam Inc. (Cambridge, MA). Goat anti-rat endothelial nitric oxide synthase (eNOS), Bax primary antibody, mouse anti-rat Bcl-2 primary antibody (Santa Cruz), mouse anti-goat secondary antibody, and rabbit anti-mouse secondary antibody were provided by Santa Cruz Biotechnology (Dallas, TX).
6
Protein Expression Analysis of TGF-β1 and SMAD3
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Western blots were performed to determine the protein expression of transforming growth factor- β1 (TGF-β1) and SMAD3. The samples were homogenized in RIPA buffer and proteinase inhibitors, and then centrifuged; the supernatant was collected, and its protein content was estimated using the Bradford protein assay kit (BioBasic, Markham, ON, Canada). Sixty μg protein was subjected to 10% SDS/PAGE followed by electro-transfer to nitrocellulose membranes. The membranes were incubated with mouse anti-TGF-β1 (Santa Cruz, CA, USA, Cat. No. sc-52893), mouse anti-SMAD3 (Santa Cruz, CA, USA; Cat. No. sc-101154), and mouse anti-β-actin (Santa Cruz, CA, USA; Cat. No. sc-8432) overnight at 4 °C. Following washing, the membranes were probed with rabbit anti-mouse secondary antibody (Santa Cruz, CA, USA, Cat. No. SC-358914) and then developed using the BCIP/NBT substrate detection kit (GeneMed Biotechnologies, Torrance, CA, USA). Protein bands were visualized using the ECL-Plus identification system (Amersham Life Sciences, Little Chalfont, Buckinghamshire, UK), according to the manufacturer’s instructions. Positive immunoreactive bands were quantified densitometrically and compared with the control.
7
Western Blot Analysis of RsbL Protein Expression
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The protein content of each sample was equalized to 0.55 mg ml−1, and 14 μl of each sample was separated by SDS-PAGE and transferred to a PVDF membrane. Western blot analysis was performed using anti-RsbL primary antibodies raised in rabbits (kindly provided by Jörgen Johannsson, Umeå University, Sweden) and mouse anti-rabbit secondary antibody (Santa Cruz Biotechnology). Blots were imaged using a chemiluminescent substrate (Amersham) on a LICOR Odyssey®Fc Imaging System (LI-COR Biosciences). Image Studio (LI-COR Biosciences) was used to process and analyze the image.
8
Extracellular Vesicle Protein Analysis
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Cells and EVs were lysed in RIPA buffer (50 mM Tris, pH 8.0, 0.6 M NaCl, 4% Triton X-100, 2% sodium deoxycholate, 0.1% SDS). Ten micrograms of cellular and EV total protein were applied per lane and separated by 10% SDS-PAGE. Proteins were electroblotted onto nitrocellulose membranes and stained with Ponceau S solution to check the protein loading. The membranes were destained, blocked with 10% (w/v) fat-free milk and then incubated with the primary antibodies: CD9 (Santa Cruz, Germany) (1:500), ALIX (Santa Cruz, Germany) (1:1000), TSG101 (Abcam, UK) (1:1000), β-actin (Abcam, UK) (1:4000) and Calnexin (Abcam, UK) (1:1000). After washing, the membranes were incubated with peroxidase - conjugated rabbit anti-mouse secondary antibody (Santa Cruz, Germany) (1:2000) or mouse anti-rabbit secondary antibody (Santa Cruz, Germany) (1:2000), washed and processed with ECL Select Western Blotting Detection Reagents (GE Healthcare, USA) according to manufacturer’s instructions.
9
Quantifying Soluble RANKL Levels
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Flat-bottom 96-well polycarbonate plates were coated at 4°C overnight with 50 μL/well cell culture supernatants diluted 1:1 in carbonate coating buffer (0.1 M Na2CO3, 0.1 M NaHCO3, pH=9.5). Standard curves were obtained with serial dilutions of purified recombinant human RANKL (Merck-Millipore) or recombinant mouse RANKL (Peprotech). After blocking with PBS supplemented with 1% W/V BSA, plates were incubated with biotin-conjugated goat anti-human RANKL (Merck-Millipore) or rabbit anti-mouse RANKL (Peprotech, USA) for 1 h at RT. Then, plates were washed with PBS-0.025% V/V Tween-20 and incubated at RT with Streptavidin-HRP-labeled secondary antibody (Invitrogen) or with a mouse anti-rabbit secondary antibody (Santa Cruz Biotechnology, Inc) for 30 min. The plates were washed, then the TMB substrate (Thermo Scientific, Inc) was added, and signal was measured using a microplate reader. All samples were run in triplicates.
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