Antibody Screening for Immunoblotting and Immunohistochemistry

Antibodies used for immunoblotting were as follows: HSC70 (# sc-7298) and TRPC1 (# sc-133076) from Santa Cruz Biotechnology; STIM1 (# ab57834) from Abcam; Orai1 (# O8264) and beta-actin (# SAB1305567) from Millipore-Sigma; Caveolain-1 (#610057) from BD Biosciences. In-house generated Protein G-purified rabbit polyclonal rabbit anti-EHD2 antisera has been described previousl (George et al., 2007 (link)). The horseradish peroxidase (HRP)-conjugated Protein A or HRP-conjugated rabbit anti-mouse secondary antibody for immunoblotting were from Invitrogen. The alpha smooth muscle actin (# ab7817), cytokeratin 8 (#53280), cytokeratin 18 (# 133263), Ki67 (# ab16667) antibodies for immunohistochemistry (IHC) and immunofluorescence (IF) staining were from Abcam. Thapsigargin (# T7459) and Fluo 4AM (# 14201) were from Thermo Fisher Scientific. Cyclopiazonic acid (# C1530) and D-luciferin (#L9504) were from Millipore Sigma. SKF96365 (cat. # S7999), SOCE inhibitor CM4620 (# S6834) from SelleckChem, Matrigel (# 356230) from Corning, and Isoflurane (# 502017) from MWI Animal Health.

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Luan H., Bielecki T.A., Mohapatra B.C., Islam N., Mushtaq I., Bhat A.M., Mirza S., Chakraborty S., Raza M., Storck M.D., Toss M.S., Meza J.L., Thoreson W.B., Coulter D.W., Rakha E.A., Band V, & Band H. (2023). EHD2 overexpression promotes tumorigenesis and metastasis in triple-negative breast cancer by regulating store-operated calcium entry. eLife, 12, e81288.

Publication 2023

HSC70 TRPC1 STIM1 Orai1 beta-actin alpha smooth muscle actin cytokeratin 8 cytokeratin 18 Thapsigargin Fluo 4AM Cyclopiazonic acid D-luciferin Matrigel

Alpha actin Animal Anti antibody Antibodies Antisera Beta actin Cm4620 Cyclopiazonic acid Cytokeratin 18 Cytokeratin 8 Fluo Horseradish peroxidase Immunofluorescence Immunohistochemistry Isoflurane Luciferin Matrigel Mouse Protein a Protein g Rabbit Skf96365 Smooth muscle Stim1 Thapsigargin

Corresponding Organization :

Other organizations : University of Nebraska Medical Center, Nottingham University Hospitals NHS Trust

Top 5 similar protocols

Variable analysis

independent variables

Thapsigargin
Cyclopiazonic acid
SKF96365
SOCE inhibitor CM4620

dependent variables

Protein expression levels of HSC70, TRPC1, STIM1, Orai1, beta-actin, Caveolain-1, EHD2, alpha smooth muscle actin, cytokeratin 8, cytokeratin 18, Ki67
Calcium signaling measured by Fluo 4AM fluorescence

control variables

Protein G-purified rabbit polyclonal rabbit anti-EHD2 antisera
Horseradish peroxidase (HRP)-conjugated Protein A or HRP-conjugated rabbit anti-mouse secondary antibody for immunoblotting
D-luciferin for bioluminescence imaging
Isoflurane for anesthesia
Matrigel for cell culture

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