Many commercially available antibodies for detection of CHOP expression as a marker of endoplasmic reticulum stress fail specificity evaluation.
Endoplasmic reticulum (ER) stress contributes to beta cell death in type 2 diabetes (T2DM). ER stress is characterized by increased level of ER stress markers such as C/EBP homologous protein (CHOP). Activation of CHOP leads to its translocation into the nucleus, where it induces cell death. We previously reported nuclear CHOP in pancreatic sections from T2DM, but not T1DM, and in human islet amyloid polypeptide (IAPP) transgenic rodent pancreatic sections. These studies underscore the importance of studying nuclear CHOP. We have observed inconsistency in the detection of CHOP antibodies reported in the literature and also in our own experiments. To investigate the specificity of CHOP antibodies, we first induced ER stress by tunicamycin in rat insulinoma (INS) cells and prepared nuclear and cytoplasmic fractions. Then we examined CHOP expression by Western blotting and immunocytochemistry using seven commercially available CHOP antibodies in INS cells and human IAPP (h-IAPP) transgenic rodent pancreatic tissue. These studies show that three commercially available CHOP antibodies out of seven tested were non-specific. In conclusion, we give recommendations for CHOP antibody selection and methods to verify CHOP antibody specificity. Also, we propose that the authors report the catalog and lot numbers of the CHOP antibodies used.
Haataja
Leena
L
Larry Hillblom Islet Research Center, UCLA David Geffen School of Medicine, Los Angeles, CA 90024-2852, USA.
Gurlo
Tatyana
T
Huang
Chang-Jiang
CJ
Butler
Peter C
PC
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D015415:Biomarkers / Q000032:analysis; D045744:Cell Line, Tumor; D004721:Endoplasmic Reticulum / Q000276:immunology / Q000378:metabolism* / Q000473:pathology*; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D010179:Pancreas / Q000166:cytology / Q000378:metabolism; D051381:Rats; D051742:Transcription Factor CHOP / Q000276:immunology / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology
D000906:Antibodies; D015415:Biomarkers; D014415:Tunicamycin; D051742:Transcription Factor CHOP