Journal: American Journal of Physiology - Renal Physiology

Article Title: Pharmacological and genetic inhibition of downstream targets of p38 MAPK in experimental nephrotic syndrome

doi: 10.1152/ajprenal.00207.2017

Figure Lengend Snippet: ADR-induced injury induced and activated small heat shock proteins (HSPs), primarily in an MK2-dependent manner. Total protein was extracted from renal cortexes isolated from control and ADR-injured mice on day 21 from each genotype and phosphorylated and total forms of HSPB1, HSPB8, glucose-regulated protein 78 (GRP78), and GAPDH were detected using their respective antibodies. A: representative Western blots of total and phosphorylated forms of selected proteins. B: densitometry analyses of protein induction/phosphorylation in the renal cortexes of at least 3 mice from each genotype. Statistical differences: *P < 0.05, control vs. ADR; #P < 0.05, WT-ADR treated vs. KO-ADR treated.

Article Snippet: The other primary antibodies used were anti-HSPB1 (StressMarq, Victoria, BC), anti-HSPB8 mouse monoclonal (Abcam, Cambridge, MA), anti-GRP78 rabbit monoclonal (StressMarq), and anti-GAPDH mouse monoclonal (Millipore, Billerica, MA).

Techniques: Isolation, Western Blot

Journal: Biology Open

Article Title: Endoplasmic reticulum stress-induced cellular dysfunction and cell death in insulin-producing cells results in diabetes-like phenotypes in Drosophila

doi: 10.1242/bio.046524

Figure Lengend Snippet: Induction of UPR observed in wing imaginal discs and IPCs with ectopic Hsc70-3 DN expression. (A–B) Expression of Xbp1-GFP generated by ER stress-dependent splicing of xbp1*-GFP mRNA in wing imaginal discs. Phase contrast (A,B) and fluorescence (A′,B′) micrographs of wing imaginal discs. (A,A′) Control wing disc ( Bx>xbp1*-GFP ). (B,B′) Wing disc expressing a dominant-negative form of Hsc70-3 in the wing pouch region (arrow) ( Bx>hsc70-3 DN , xbp1*-GFP ). (C–E) Fluorescence micrograph of wing discs stained with DAPI (white). (C′–E′) Immunostaining of the wing discs with an anti-GRP78 antibody. (D″) Immunostaining of the wing disc with anti-HA antibody. (C,C′) Fluorescence micrograph of a control wing imaginal disc ( Bx-Gal4/+ ). (D–D″) Wing imaginal disc expressing control Hsc70-3 in the wing pouch region of the imaginal disc ( Bx>hsc70-3 ). (E,E′) Wing imaginal disc expressing a dominant-negative form of Hsc70-3 in the same region ( Bx>hsc70-3 DN ). Anti-GRP78 immunostaining is shown in white. Note that more intense immunofluorescence was observed exclusively in areas expressing Hsc70-3 DN , but not the control protein. (A–F) Relative intensity of anti-GRP78 immunostaining in wing imaginal discs. Immunofluorescence signal intensity in each wing imaginal disc with the control Hsc70-3 ( n =31) or Hsc70-3 DN ( n =25) expression was calculated and normalized to the control value, which was set as 1.0 ( Bx-Gal4/+ ) ( n =25; n.s., not significant, P >0.05; *** P <0.001, Student's t -tests). Error bars represent s.e.m. (G–I) Anti-GRP78 immunostaining of IPCs expressing GFPnls in brains from third-instar larvae. (G) Control IPCs ( ilp2>GFPnls ), (H) IPCs expressing the control Hsc70-3 ( ilp2>hsc70-3, GFPnls ), (I) IPCs expressing Hsc70-3 DN ( ilp2>hsc70-3 DN , GFPnls ). Anti-GRP78 immunostaining is colored in red (G–I; white in G′–I′). Nuclei of IPCs visualized by GFPnls expression are colored green (G–I; white in G″–I″). Arrows in H′ and H″ indicate positions of IPC cells. Note that remarkably higher immunostaining signal was observed in IPCs expressing Hsc70-3 DN , but not the control protein. (J) Relative intensities of anti-GRP78 immunostaining in larval IPCs. Immunofluorescence signal intensities in each IPC expressing Hsc70-3 ( n =25) or Hsc70-3 DN ( n =21) were calculated and normalized to the control value of 1.0 ( ilp2>GFPnls ) ( n =21, * P <0.05, *** P <0.001, Student's t -test). Error bars represent s.e.m. Scale bars: (A–E) 100 µm, (G–I) 50 µm.

Article Snippet: The following primary antibodies were used at the dilution described; rabbit anti-β-galactosidase (MP Biomedicals, #55976) at 1:1000, rabbit anti-GRP78 (Bip) (StressMarq Biosciences Inc., Cadboro Bay, Victoria, Canada) that could recognize Hsp70 family proteins including Hsc70-3 in Drosophila at 1:500, rabbit Cleaved Caspase-3 (Asp175) (#9661, Cell Signaling, Danvers, Massachusetts, USA) at 1:200 for larval brain immunostaining and at 1:150 for wing disc immunostaining, rabbit anti-Cleaved Drosophila Dcp-1 (Asp216) (Cell Signaling, antibody #9578) at 1:500, and rabbit anti-phospho-SAPK/JNK (pThr183, pTyr185) (Calbiochem, La Jolla, CA, USA) at 1:200.

Techniques: Expressing, Generated, Fluorescence, Dominant Negative Mutation, Staining, Immunostaining, Immunofluorescence