A rabbit polyclonal antibody against the peptide corresponding to amino acids 339 to 359 (CDGLNDSSKQGYSQETKRLKRT) of human PTAR1 primary sequence was custom made by YenZyme antibodies, LLC, CA 94080. The specificity of the antibody was validated by silencing PTAR1 in primary fibroblasts NHFs and HEK293T cell lines (Supplementary Fig. 1A ). Antibodies were from Abcam (FNTA, FNTB), Abnova (PGGT1B), Bethyl (RabGGTB), Aviva Biosystems (RabGGTA), Sigma (anti-FLAG M2, anti-FLAG, β-actin), Santa Cruz Biotechnology (SKP1), and Covance (anti-HA). A polyclonal rabbit antibody to FBXL2 was generated using a peptide against FBXL2 as previously described27 (link). Human cDNA ORFs for PTAR1, FNTA, and RabGGTA were purchased from Origene. GFP-tagged cDNA of RAB1a, RAB7a, RAB11, RAB34, and RAB35 were obtained from Addgene. The variously tagged-genes and mutants were generated in pcDNA3 and pEGFPC1 vectors either by sub-cloning or site-directed mutagenesis methodologies, as described.
Protocol detail
Validation of PTAR1 antibody specificity
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Actin
Amino acids
Antibodies
Antibody
Antibody specificity
Cdna
Cell lines
Cloning vectors
Fibroblasts
Fnta
Genes
Human
Orfs
Peptide
Pggt1b
Rabbit
Site directed mutagenesis
Skp1
Corresponding Organization :
Other organizations : New York University, University of Washington, Howard Hughes Medical Institute
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Variable analysis
independent variables
- Silencing PTAR1 in primary fibroblasts NHFs and HEK293T cell lines
- Specificity of the PTAR1 antibody
- Antibodies from Abcam (FNTA, FNTB), Abnova (PGGT1B), Bethyl (RabGGTB), Aviva Biosystems (RabGGTA), Sigma (anti-FLAG M2, anti-FLAG, β-actin), Santa Cruz Biotechnology (SKP1), and Covance (anti-HA)
- Polyclonal rabbit antibody to FBXL2 generated using a peptide against FBXL2
- Human cDNA ORFs for PTAR1, FNTA, and RabGGTA purchased from Origene
- GFP-tagged cDNA of RAB1a, RAB7a, RAB11, RAB34, and RAB35 obtained from Addgene
- Variously tagged-genes and mutants generated in pcDNA3 and pEGFPC1 vectors either by sub-cloning or site-directed mutagenesis methodologies
- Silencing PTAR1 in primary fibroblasts NHFs and HEK293T cell lines to validate the specificity of the PTAR1 antibody
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