Human WT pED-FLAG-LMAN1, pED-FLAG-LMAN1 DCRD (R44-E296), pED-FLAG-LMAN1 DHelix (G271-N457), and pED-FLAG-LMAN1 N156A were reported previously.63 (link) Transient transfection of HEK293T cells or NIH/3T3 was performed using calcium phosphate transfection. Cell lysates for western blot were obtained using cell lysis buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM β-glycerophosphate, 1 mM PMSF, 1 mM NaVO4, 10 mM calyculin A, protease inhibitor cocktail]. Cell lysates were ran on 10% percent bis/acrylamide gels using a Mini-Protean Tetra cell apparatus and transferred to nitrocellulose membranes using a Criterion blotter (all from Bio-Rad; Hercules, California) (Cat# 1658000, Cat# 1620112, Cat# 1704071). LMAN1 was detected using an α-LMAN1 antibody (Cell Signaling; Danvers, MA) (Cat# 13947) or using an α-FLAG antibody (MilliporeSigma; Burlington, MA) (Cat# F1804). Total protein was detected using stain free gels (Bio-Rad; Hercules, California) (Cat# 1610182).
Tetra cell apparatus
Manufactured by Bio-Rad
The Tetra cell apparatus is a piece of laboratory equipment designed for performing gel electrophoresis. It is used to separate and analyze biomolecules such as proteins, DNA, or RNA based on their size and charge. The core function of the Tetra cell apparatus is to provide a controlled environment for the electrophoretic separation process.
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2 protocols using tetra cell apparatus
1
LMAN1 Protein Expression and Analysis
2
Western Blot Analysis of TRPV5 and TRPV6
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Western blot analyses were carried out on protein extracts (P1 and S1) as previously described 18 (link),20 (link). Aliquots of 60 μg of proteins were mixed with SDS sample buffer (Laemmli solution) and heated at 100°C for 5 min. Proteins were run on 8% SDS-PAGE gels, and transferred onto nitrocellulose membranes (0.2 μm) by using 100 V for 30 min. (Tetra cell apparatus, Bio-Rad). Anti-TRPV5 and anti-TRPV6 goat polyclonal antibodies (K-17 and L-15, respectively, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) were used at dilution of 1:200. After incubation with appropriate secondary antibody, immunoreactions were visualized by using ECL detection system. The chemiluminescent images were acquired by LAS4010 (GE Health Care Europe, Uppsala, Sweden).
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