Journal: Molecular and cellular endocrinology

Article Title: Defective endoplasmic reticulum export causes proinsulin misfolding in pancreatic β cells

doi: 10.1016/j.mce.2019.110470

Figure Lengend Snippet: A. Western blotting analysis of total cellular and secreted proinsulin in MIN6 cells infected with adenovirus expressing GFP (Knowler et al.), Sar1A wild-type (WT), Sar1A H79G (H79G) or Sar1A T39N (T39N) mutants. B. Quantification of the secreted proinsulin in the culture medium. Secreted proinsulin was normalized to the corresponding total cellular proinsulin levels. Statistical analysis: Student’s t-test, * P≤ 0.05. Error bars indicate standard error of the mean (SEM). C. Immunofluorescent images of control (Knowler et al.), Sar1A wild-type and mutant adenovirus-infected MIN6 cells. Red: immunostaining of proinsulin. D. Western blotting of MIN6 cells infected with GFP control (Knowler et al.), wild-type (WT), and mutant Sar1A (H79G,T39N) adenovirus for 24 hours. The total proteins were resolved on 4–12% gradient gel under reduced (right) or non-reduced (left) conditions and blotted with anti-proinsulin, Sar1A and β-actin antibodies. The proinsulin-specific antibody (Novus Biologicals) not only recognized proinsulin monomer in both the reduced and non-reduced forms but also detected the disulfide linked proinsulin complexes (DLPC) in the non-reduced samples. E. Quantifications of the misfolded proinsulin expressed as the fold change in total intensity of disulfide-linked proinsulin complex compared to that of control. Statistical analysis: Student’s t-test. * P≤0.05. Error bars indicate standard error of the mean (SEM). F. Western blotting analysis of MIN6 cells treated with or without 5μm Brefeldin (BFA) for 5 hrs. The total proteins were resolved on 4–12% gradient gel under reduced (right) or non-reduced (left) conditions and blotted with a proinsulin-specific antibody (Novus Biologicals).

Article Snippet: Materials The following antibodies were used in our study: proinsulin (Novus Biologicals); C/EBP-homologous protein (CHOP), Sar1B, and β-actin (GeneTex); BiP (BD Bioscience); Sar1A, and PDI (Proteintech); p-eIF2α and eIF2α (Cell Signaling Technology); guinea pig polyclonal anti-insulin antibody was custom made; and the Sec31A polyclonal antibody was a gift from Dr. Fred Gorlick at Yale University.

Techniques: Western Blot, Infection, Expressing, Control, Mutagenesis, Immunostaining

Journal: Molecular and cellular endocrinology

Article Title: Defective endoplasmic reticulum export causes proinsulin misfolding in pancreatic β cells

doi: 10.1016/j.mce.2019.110470

Figure Lengend Snippet: A. MIN6 cells were transfected with scramble siRNA or equally mixed siRNAs against INS 1 and INS 2. 48 hours later, the transfected cells were infected with GFP control, wild-type (WT), or mutant Sar1A (H79G,T39N) adenovirus for another 24 hours before harvested for Western blotting analysis using indicated antibodies. B. Aliquots of the same samples as in A were prepared and analyzed in non-reduced condition. Proinsulin-specific antibody was used to detect DLPC (disulfide linked proinsulin complex). C. Quantifications of the misfolded proinsulin expressed as the fold change in total intensity of disulfide linked proinsulin complex (DLPC) compared to that of control. Statistical analysis: Student’s t-test. * P ≤ 0.05. Error bars indicate standard error of the mean (SEM). D, E, and F. Quantifications of fold changes in BiP, p-eIF2α, and CHOP protein levels upon different treatments compared to that of control. While BiP and CHOP expressions were normalized with actin, p-eIF2α was normalized with total eIF2α. Statistical analysis: Student’s t-test. * P≤0.05. Error bars indicate standard error of the mean (SEM)

Article Snippet: Materials The following antibodies were used in our study: proinsulin (Novus Biologicals); C/EBP-homologous protein (CHOP), Sar1B, and β-actin (GeneTex); BiP (BD Bioscience); Sar1A, and PDI (Proteintech); p-eIF2α and eIF2α (Cell Signaling Technology); guinea pig polyclonal anti-insulin antibody was custom made; and the Sec31A polyclonal antibody was a gift from Dr. Fred Gorlick at Yale University.

Techniques: Transfection, Infection, Control, Mutagenesis, Western Blot

Journal: Molecular and cellular endocrinology

Article Title: Defective endoplasmic reticulum export causes proinsulin misfolding in pancreatic β cells

doi: 10.1016/j.mce.2019.110470

Figure Lengend Snippet: A. Western blotting analysis of proinsulin using a specific C-A junction antibody. This antibody not only recognized proinsulin monomer in both reduced and non-reduced conditions, but also detected the disulfide linked proinsulin complex (DLPC) in the non-reduced samples. 293T cells were transfected with human Akita(proinsulin)-Myc or human WT (proinsulin)-Myc plasmids for 48 hours. The total proteins were resolved in 4–12% gradient gel under reduced (right) or non-reduced (left) conditions and blotted with anti-proinsulin(C-A junction). B. Immunofluorescent staining of a COPII coat protein-Sec31A, in dispersed human islet cells treated with wild type (WT) or mutant Sar1A (H79G,T39N) adenoviruses. C. Western blotting analysis of isolated human islets infected with GFP control, wild-type (WT), and mutant Sar1A (H79G, T39N) adenovirus. The total proteins were separated by SDS-PAGE under non-reduced (upper) or reduced (bottom) conditions and then blotted with anti-proinsulin (C-A junction), anti-Sar1A or anti-β-actin antibody. D. Quantifications of the misfolded proinsulin expressed as the fold change in total intensity of disulfide linked proinsulin complex (DLPC) compared to that of control. Statistical analysis: Student’s t-test. * P≤0.05. Error bars indicate standard error of the mean (SEM).

Article Snippet: Materials The following antibodies were used in our study: proinsulin (Novus Biologicals); C/EBP-homologous protein (CHOP), Sar1B, and β-actin (GeneTex); BiP (BD Bioscience); Sar1A, and PDI (Proteintech); p-eIF2α and eIF2α (Cell Signaling Technology); guinea pig polyclonal anti-insulin antibody was custom made; and the Sec31A polyclonal antibody was a gift from Dr. Fred Gorlick at Yale University.

Techniques: Western Blot, Transfection, Staining, Mutagenesis, Isolation, Infection, Control, SDS Page

Journal: Molecular and cellular endocrinology

Article Title: Defective endoplasmic reticulum export causes proinsulin misfolding in pancreatic β cells

doi: 10.1016/j.mce.2019.110470

Figure Lengend Snippet: A.MIN6 cells treated with either wild type (WT) or mutant Sar1A(H79G) adenovirus were lysed to immunoprecipitate proinsulin and its interacting proteins using a proinsulin specific antibody and magnetic protein A/G beads. The immunoprecipitants were then analyzed by Western blotting using antiproinsulin, anti-BiP, and anti-PDI antibodies (left). Total lysates (10% of the input for the immunoprecipitation) were also analyzed by Western blotting using the indicated antibodies. B and C. Quantifications of the fold changes in the amount of BiP or PDI in the total lysis between the wild type Sar1A(WT) and mutant Sar1A(H79G) expressing cells. Statistical analysis: Student’s t-test. *P≤0.05. Error bars indicate standard error of the mean (SEM). D and E. Quantifications of the fold changes in the amount of BiP or PDI in the immunoprecipitants between the wild type Sar1A(WT) and mutant Sar1A(H79G) expressing cells. Statistical analysis: Student’s t-test. * P≤0.05. Error bars indicate standard error of the mean (SEM).

Article Snippet: Materials The following antibodies were used in our study: proinsulin (Novus Biologicals); C/EBP-homologous protein (CHOP), Sar1B, and β-actin (GeneTex); BiP (BD Bioscience); Sar1A, and PDI (Proteintech); p-eIF2α and eIF2α (Cell Signaling Technology); guinea pig polyclonal anti-insulin antibody was custom made; and the Sec31A polyclonal antibody was a gift from Dr. Fred Gorlick at Yale University.

Techniques: Mutagenesis, Western Blot, Immunoprecipitation, Lysis, Expressing

Journal: Molecular and cellular endocrinology

Article Title: Defective endoplasmic reticulum export causes proinsulin misfolding in pancreatic β cells

doi: 10.1016/j.mce.2019.110470

Figure Lengend Snippet: A. Western blotting of MIN6 cells transfected with scramble siRNA (control), Sar1A or Sar1B siRNA, or equally mixed of Sar1A and Sar1B siRNA. The total proteins were resolved in 4–12% gradient gel under non-reduced (upper) or reduced (bottom) conditions and blotted with anti-proinsulin, anti-insulin, anti-Sar1A, anti-Sar1B or anti-β-actin. B. Quantifications of the misfolded proinsulin expressed as the fold change in total intensity of disulfide-linked proinsulin complex (DLPC) compared to that of control. Statistical analysis: Student’s t-test. * P≤0.05. Error bars indicate standard error of the mean (SEM). C. Immunofluorescent staining of MIN6 cells transfected with scramble siRNA or with equally mixed Sar1A and Sar1B siRNAs for 3 days: proinsulin (red), PDI (green). D. Immunofluorescent staining of MIN6 cells transfected with scramble siRNA or with equally mixed Sar1A and Sar1B siRNAs for 3 days: Sec31A (red).

Article Snippet: Materials The following antibodies were used in our study: proinsulin (Novus Biologicals); C/EBP-homologous protein (CHOP), Sar1B, and β-actin (GeneTex); BiP (BD Bioscience); Sar1A, and PDI (Proteintech); p-eIF2α and eIF2α (Cell Signaling Technology); guinea pig polyclonal anti-insulin antibody was custom made; and the Sec31A polyclonal antibody was a gift from Dr. Fred Gorlick at Yale University.

Techniques: Western Blot, Transfection, Control, Staining