Murine cerebrovascular endothelial cells underwent Western blotting as previously described by Yin et al.9 (link). Briefly, the cells were lysed via lysis buffer to isolate the total protein. Equal protein amounts were subjected to SDS-PAGE followed by transfer to polyvinyldifluoride (PVDF) membranes. PVDF membranes were then blocked in a blocking solution (5% non-fat milk in Tris-buffered saline) followed by incubation with the following primary antibodies for two hours at room temperature: anti-PPARγ (1:200; catalog # sc-7273, Santa Cruz Biotechnology), anti-CD36 (1:200; catalog # sc-9154, Santa Cruz Biotechnology), and β-actin (1:500; catalog # sc-47778, Santa Cruz Biotechnology). The membranes were then incubated for one hour at room temperature with the appropriate alkaline phosphatase-conjugated secondary antibodies (Promega). The ProtoBlot AP System (Promega) was applied to assess the color reaction according to the kit’s instructions.
Sc 7273
Manufactured by Santa Cruz Biotechnology
Sourced in United States, China
Sc-7273 is a laboratory equipment product offered by Santa Cruz Biotechnology. It is a high-quality instrument designed for specific research applications. The core function of Sc-7273 is to perform tasks essential for scientific investigations, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Pparγ, by Santa Cruz Biotechnology (4 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Sc 61, by Santa Cruz Biotechnology (2 mentions)
Sc 13067, by Santa Cruz Biotechnology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Anti pparγ, by Santa Cruz Biotechnology (1 mentions)
Anti cd36, by Santa Cruz Biotechnology (1 mentions)
Sc 47778, by Santa Cruz Biotechnology (1 mentions)
Sc 9154, by Santa Cruz Biotechnology (1 mentions)
Alkaline phosphatase conjugated secondary antibody, by Promega (1 mentions)
Ab2064, by Abcam (1 mentions)
Ab44718, by Abcam (1 mentions)
Ab28829, by Abcam (1 mentions)
Ab203294, by Abcam (1 mentions)
Proteinase inhibitor cocktail, by Merck Group (1 mentions)
Ab13556, by Abcam (1 mentions)
Enhanced chemiluminescence reagent, by Bio-Rad (1 mentions)
Goat anti rabbit igg h l, by Jackson ImmunoResearch (1 mentions)
Goat anti mouse igg h l, by Jackson ImmunoResearch (1 mentions)
28 protocols using sc 7273
1
Murine Endothelial Cell Protein Expression
2
Protein Expression Profiling of Signaling Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells or tissues were harvested at indicated times and homogenized in ice-cold suspension buffer supplemented with a proteinase inhibitor cocktail (Sigma-Aldrich). Protein concentrations were determined using the BCA Protein assay kit (Thermo Scientific, Waltham, MA). Equal amounts of protein were fractionated by SDS polyacrylamide gels, followed by immunoblotting with the following primary antibodies: NF-κB Pathway Sampler Kit (diluted at 1:1000, 9936, CST, MA), phospho-MAPK Family Antibody Sampler Kit (diluted at 1:1000, 9910, CST, MA), MAPK Family Antibody Sampler Kit (diluted at 1:1000, 9926, CST, MA), phospho-Stat6 antibody (Rabbit polyclonal, diluted at 1:1000, ab28829, Abcam), Stat6 antibody (Rabbit polyclonal, diluted at 1:1000, ab44718, Abcam), TRAF6 antibody (Rabbit polyclonal, diluted at 1:1000, ab94720, Abcam), TLR4 antibody (Rabbit polyclonal, diluted at 1:1000, ab13556, Abcam), CD14 antibody (Rabbit polyclonal, diluted at 1:1000, ab203294, Abcam), Myd88 antibody (Rabbit polyclonal, diluted at 1:1000, ab2064, Abcam), PPARγ antibody (Rabbit monoclonal (C26H12), diluted at 1:1000, sc-7273, Santa Cruz Biotechnology). Membranes were then incubated with peroxidase-conjugated secondary antibody, and specific bands were detected with a Bio-Rad (Hercules, CA) imaging system. Original blots were provided in Supplementary Fig. 8 .
3
Skeletal Muscle Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were harvested from the rat’s soleus skeletal muscle (50 mg) using cell lysis buffer (10 mM HEPES at pH 7.9, 10 mM KCI, 1.5 mM MgCl2, and 1 mM dithiothreitol). The total proteins (45 µg/mL) were separated in 10% polyacrylamide gels with SDS and a PVDF membrane (Bio-Rad) and transferred into a Transblot Turbo transference system (Bio-Rad). The membranes were preincubated for 1.5 h at ambient temperature with 8% nonfat milk in tris buffered solution (100 mM NaCl, 20 mM Tris; pH 7.6) supplemented with 0.05% Tween 20. Next, they were incubated with specific primary antibodies (INSR: sc-09 Santa Cruz Biotechnology 1:500; Phospho-IRS1 (Tyr612): 44-816G Invitrogen 1:500; PI3K-C2α: sc-365290 Santa Cruz Biotechnology 1:500; PPAR-γ: sc-7273 Santa Cruz Biotechnology 1:500; PPAR-α: PA1-822A Invitrogen 1:500; GLUT-4: G4048 Sigma-Aldrich 1:500 and, β-actin: sc-47778 Santa Cruz Biotechnology 1:500) overnight at four °C and then with a horseradish peroxidase-conjugated secondary antibody (Goat Anti-Rabbit IgG H+L: 111-035-003 Jackson ImmunoResearch 1:1000; Goat Anti-Mouse IgG H+L: 115-035-003 Jackson ImmunoResearch 1:1000) at ambient temperature. Bands were visualized with enhanced chemiluminescence reagents (Bio-Rad, Hercules, CA, USA). Band densities were quantified using ImageJ software (version 8, NIH).
4
Quantification of PPARγ Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Adipose tissue was homogenized in ice-cold RIPA buffer with protease inhibitors (Protease inhibitor Cocktail Set III, Calbiochem, La Jolla, CA). Protein concentration was quantified using a BCA protein assay (Thermo Fisher Scientific, Hanover Park, IL, USA). For immunoblots, 30 µg of protein sample was separated by 10% SDS-PAGE and transferred to a PVDF membrane (MilliporeSigma, Burlington, MA, USA). Membranes were blocked in 5% nonfat dry milk (w/v) and incubated with a primary antibody against PPARγ (Santa Cruz Biotechnology, sc-7273, Santa Cruz, CA, USA) or GAPDH (MAB374, Sigma-Aldrich, St. Louis, MO, USA), followed by IgG-horseradish peroxidase-conjugated secondary antibody. Blots were developed using Amersham ECL Prime Detection Reagent (VWR, West Chester, PA, USA).
5
Sericin hydrogel modulates osteogenic differentiation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The adenovirus-infected cells were seeded in 6-well plates and treated with WT sericin hydrogel or 3.44 × 10−3 μg/mL PDGF-BB sericin hydrogel. Cells were harvested at 24 h, 48 h, 9 days or 11 days after treatment. Cell lysates were separated by 10% SDS-PAGE (Beyotime) and transferred to PVDF (Polyvinylidene fluoride) membrane (Bio-Rad, CA, USA). The membrane was pre-blocked with 5% bovine serum albumin buffer for 60 min, probed with the primary antibody (1:1,000), washed three times with Tris-buffered saline containing 0.1% Tween-20 and then incubated with horseradish peroxidase-conjugated secondary antibodies. Primary antibodies for GAPDH (sc-32233), osteopontin (OPN) (sc-10593), Runx2 (sc-12488), osteocalcin (OCN) (sc-365797), peroxisome proliferators activated receptor-γ (PPARγ) (sc-7273), CCAAT/enhancer binding protein α (C/EBP α) (SC-61), Smad 1/5/9 (AF0614), p-Smad 1/5/9 (AF8313), Stat3 (60199-1) and p-Stat3 (SC-8059) were purchased from Santa Cruz Biotechnology (China). Assays were done in triplicate.
6
Molecular Profiling of Muscle Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gastrocnemius and hearts lysates (obtained by centrifugation of homogenates) were separated by SDS-PAGE, transferred to nitrocellulose membranes and probed using antibodies against MyoD, Myogenin (M3512, M3559-Dako), Desmin, Collagen type-III, α-SMA (D1033, C7805, A5228-Sigma-Aldrich), PGC1α, β-ATPase (AB3242, MAB3494-Millipore), TFAM, Tom20, CSQ2, fsTn-I, VEGF, VE-Cadherin, PECAM, SDHA and PPARγ (sc-23588, sc-11415, sc-390999, sc-377382, sc-7269, sc-9989, sc-46694, sc-377302, sc-7273-Santa Cruz Biotechnology), CoxIV (Ab14744-Abcam) and the appropriate secondary antibodies. α-tubulin or actin (T5168, A3853-Sigma-Aldrich) were used for normalization. Quantification was performed as previously described (Ferraro et al., 2016 (link)).
7
Immunofluorescence Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Formalin-fixed paraffin sections were deparaffinized, rehydrated and immersed in PBS. Antigen retrieval was performed with citric acid (0.01 M, pH 6.0), and non-specific sites of the sections were blocked by incubation with PBS containing 10% donkey serum and 0.2% Triton X-100 for 1 h. The sections were incubated overnight at 4 °C with primary antibodies diluted in PBS containing 0.2% Triton X-100. Immune complexes were detected by incubation for 1 h at room temperature with Alexa Fluor 488-, Alexa Fluor 594- or Alexa Fluor 647-conjugated secondary antibodies at a dilution of 1:500 in PBS. The sections were then washed three times for 5 min with PBS, exposed to DAPI (D9542, Sigma) for 5 min to stain nuclei, washed briefly with PBS, mounted with ProLong Gold antifade reagent (P36930, Invitrogen) and sealed with a cover glass (0101172 or 0101192, Marienfeld). Primary antibodies were used at dilutions of 1:500 for red fluorescent protein (600401379, Rockland, or AB8181-200, Sicgen) and Ki67 (ab16667, Abcam), 1:200 for PPARG (sc-7273, Santa Cruz Biotechnology), PDGFRA (AF1062, R&D Systems) and YAP/TAZ (8418S, Cell Signaling).
8
Adipose Tissue Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from adipose tissues or cultured cells, using RIPA lysis buffer with protease inhibitors cocktail (Selleck). Protein lysates were resolved by SDS-PAGE and transferred onto PVDF membranes. Membranes were then incubated with primary antibodies, including anti-UCP-1 (Abcam, ab23841), anti-perilipin (Abcam, ab61682), anti-APN (R&D, AF1119), anti-PPARγ (Santa Cruz, sc-7273), anti-PGC1α (Santa Cruz, sc-13067), and β-actin for normalization. Subsequently, incubation with HRP-conjugated antibodies, and then protein signals were detected with electrochemiluminescence (ECL) detection reagents.
9
Protein Expression Analysis in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The harvested cells were lysed using RIPA buffer (Sigma-Aldrich) according to the manufacturer’s instructions. The protein concentrations were measured with Pierce™ BCA Protein Assay Kit #23225 (Thermo Fisher Scientific Inc., Waltham, MA, USA). Forty μg of protein were loaded for sodium dodecylsulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. After blocking with 5% skim milk in tris-buffered saline, the membrane was incubated with mouse anti-CD36 (1:2000; TA500921, OriGene, Rockville, MD, USA), rabbit anti-IL-18 (1:1000; ab191152, Abcam, Cambridge, MA, USA), mouse anti-IL-6 (1:1000; ab9324, Abcam), and mouse anti-PPAR-γ (1:1000; sc-7273, Santa Cruz Biotechnology Inc., Dallas, TX, USA) overnight at 4 °C. The membrane was washed with tris-buffered saline and incubated with their corresponding horseradhish peroxidase-conjugated secondary antibodies (1:5000) for 1 h at room temperature. An enhanced chemiluminescence system (GE Healthcare, Little Chalfont, UK) was used to detect target proteins. The protein expression was normalized with the signal obtained with β-actin expression [35 (link)].
10
Immunoprecipitation of PPAR-γ, HDAC3, and RORα
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Phosphate-buffered saline was used to wash the cells before lysing them in lysis buffer (200 mM NaCl, 50 mM Tris-HCl, pH 8.0, and 0.5% NP40). After that, the lysates were mixed with protein A beads (CST, Waltham, MA; 70024) and incubated overnight at 4°C with primary antibodies against anti-PPAR-γ (sc-7273; Santa Cruz Biotechnology, Dallas, TX), anti-HDAC3 (ab32369; Abcam, Boston, MA), anti-RORα (ab256799; Abcam), or the corresponding normal IgG (acts as negative control). To remove unbound proteins, the beads were washed twice with lysis buffer. To elute the bound proteins, the beads were resuspended in sample buffer, boiled for 3 minutes at 95°C, and immunoblotted.
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!