Inguinal white adipose tissues were collected and fixed with 10% formalin (Fisher, Pittsburgh, PA). After paraffin imbedding, tissue samples were sectioned (5-μm) and stained for UCP1 (Sigma-Aldrich) and Pref-1 (Cell Signaling Technology, Beverly, MA). Immunohischemistry was performed with avidin-biotin elite kit (Vector Laboratories, Burlingame, CA) following the manufacturer’s protocol. For electron microscopy, fresh isolated EDL and Soleus muscles for wild-type and mCaROCK1 mice were pinned on a small plastic board at rest position and followed a fix in 2% paraformal-dehyde +2.5% glutaraldehyde in 0.1 m sodium cacodylate buffer for overnight at 4 °C. Then, samples were incubated in 1% osmium tetroxide followed by 1% uranyl acetate for 1 h. After dehydration, samples were embedded in resin with alignment of the longitudinal axis of myofibers. Thin (80–100 nm) sections were cut using an Ultracut E ultramicrotome (Leica Microsystems, Bannockburn, IL). After stained with uranyl acetate/lead citrate, sections were examined and imaged at 80 keV using Hitachi H-7500 transmission electron microscopy.
Pref 1
Manufactured by Cell Signaling Technology
Pref-1 is a laboratory product designed for use in cell biology research. It functions as a cell surface marker that can be used to identify and study preadipocyte cells, which are precursor cells that can differentiate into adipocytes (fat cells).
Automatically generated - may contain errors
Lab products found in correlation
Pparγ, by Cell Signaling Technology (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Formalin, by Thermo Fisher Scientific (1 mentions)
Ultracut e ultramicrotome, by Leica (1 mentions)
H 7500 transmission electron microscopy, by Hitachi (1 mentions)
Phosphor akt, by Cell Signaling Technology (1 mentions)
Phosphor erk, by Cell Signaling Technology (1 mentions)
Phosphor jnk, by Cell Signaling Technology (1 mentions)
Noggin, by Proteintech (1 mentions)
Hsp90, by Santa Cruz Biotechnology (1 mentions)
Phosphor p65, by Cell Signaling Technology (1 mentions)
Bmpria, by Abcam (1 mentions)
Smad4, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer system, by Santa Cruz Biotechnology (1 mentions)
Streptavidin europium conjugate, by PerkinElmer (1 mentions)
Fluostar optima microplate reader, by BMG Labtech (1 mentions)
Mouse adipokine array kit, by R&D Systems (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
5 protocols using pref 1
1
Adipose Tissue and Muscle Ultrastructure
2
Immunoblotting Analysis of Signaling Pathways
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Cells were scraped into lysis buffer containing 2% sodium dodecyl sulfate (SDS) and 50 mM Tris–HCl (pH 6.8). Lysates were quantitated and equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Proteins were then transferred onto Polyvinylidene fluoride (PVDF) membrane and immunoblotted with specific antibodies. Antibodies used were PPARγ, phosphor-p38 MAPK, p38 MAPK kinase, phosphor-Smad1/5/8, Smad1, Smad4, Pref1, phosphor-Akt, Akt, phosphor-ERK, ERK, phosphor-JNK, JNK, phosphor-p65, p65 (Cell Signaling Technology), BMPRIA (Abcam), Noggin (Proteintech), B4GalT5 (Sigma), Hsp90 (Santa Cruz Biotechnology), and C/EBPα, and 422/aP2 (obtained from the Department of Biological Chemistry at the Johns Hopkins University School of Medicine).
3
Exosomal and Adipocyte Marker Analysis
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EVs at day 0 and day 15 were probed for exosomal and adipocyte markers using high protein binding ELISA plates (Greiner Bio-One, Germany). Protein concentration of EV samples was determined using a Pierce™ BCA Protein Assay Kit (Thermo Scientific, UK) and 1 µg of EV sample was loaded into each well. EVs were allowed to settle overnight before a buffer (RIPA Lysis Buffer System; Santa Cruz, CA, USA) was added to permeabilise EVs for analysis of intravesicular EV and adipocyte markers. Exosomal markers included rabbit anti-mouse CD9, CD63, tumour susceptibility gene 101 (TSG101) (Santa Cruz) and mouse anti-mouse alix (Cell Signaling Technologies, New England BioLabs, UK). Adipocyte markers included rabbit anti-mouse FABP4, peroxisome proliferator-activated receptor γ (PPARγ), adiponectin and preadipocytes factor-1 (PREF-1) (Cell Signaling Technologies). Markers were detected using anti-mouse or anti-rabbit biotin-labelled secondary antibodies (Perkin Elmer) and a streptavidin–europium conjugate (Perkin Elmer) and analysed using time resolved fluorescence (Wallac Victor2 1420 plate reader, Perkin Elmer; and FLUOstar OPTIMA microplate reader, BMG Labtech, UK).
4
Western Blot and Adipokine Analysis of Adipogenesis
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Cells and tissues were homogenized into lysis buffer containing 2% sodium dodecyl sulfate (SDS) and 60 mM Tris·HCl (pH6.8). Lysates were quantitated and equal amounts of protein were loaded to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Antibodies used are C/EBPα and HSP90 (Santa Cruz Biotechnology), PPARγ and Pref-1 (Cell Signaling Technology).
For the adipokine array, cells supernatants were analyzed by Mouse Adipokine Array kit (R&D), and quantified by ImageJ.
For the adipokine array, cells supernatants were analyzed by Mouse Adipokine Array kit (R&D), and quantified by ImageJ.
5
Western Blot Analysis of Pref-1 and GAPDH
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For western blots, cultured cells were lysed in 2% SDS buffer plus protease and phosphatase inhibitors (Thermo Scientific, USA). The lysates of equivalent total proteins were separated on SDS-PAGE. Then proteins were transferred into PVDF membrane (Millipore) for incubation with primary/secondary antibodies. The primary antibodies used were as follows, Pref-1 (Cell Signaling Technology, Cat#2069) and GAPDH (Cell Signaling Technology, Cat#2118).
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