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

Journal: Molecular Medicine Reports

Article Title: Sulforaphane protects against oxidative stress-induced apoptosis via activating SIRT1 in mouse osteoarthritis

doi: 10.3892/mmr.2021.12251

Figure Lengend Snippet: Effect of SFN on H 2 O 2 -induced endoplasmic reticulum stress and subsequent apoptosis in mouse chondrocytes. Chondrocytes were pretreated with 50 µM SFN for 24 h and incubated for 24 h in the presence or absence of H 2 O 2 (50 µM). (A) Western blot analysis of levels of (B) CHOP and (C) GRP78 were assessed by western blotting. (D and E) Chondrocyte apoptosis was evaluated by In Situ Cell Death Detection kit (scale bar, 50 µm). (F) Quantification of ROS levels. Data are presented as the mean ± SD (n=3). ## P<0.01 vs. control. **P<0.01 vs. H 2 O 2 -alone. SFN, sulforaphane; CHOP, C/EBP-homologous protein; GRP78, glucose-regulated protein 78; ROS, reactive oxygen species.

Article Snippet: Membranes were blocked using 5% skimmed milk for 2 h at 37°C and detected overnight at 4°C with the following antibodies: CHOP (1:1,000; cat. no. 15204-1-AP; Wuhan Sanying Biotechnology), GRP78 (1:1,000; cat. no. 11587-1-AP; Wuhan Sanying Biotechnology), Bcl-2 (1:1,000; cat. no. 15071; Cell Signaling Technology, Inc.), Bax (1:1,000; cat. no. 5023; Cell Signaling Technology, Inc.), Cleaved caspase-3 (1:1,000; cat. no. 9661; Cell Signaling Technology, Inc.), SIRT1 (1:500; cat. no. 2493; Cell Signaling Technology, Inc.) and GAPDH (1:1,000; cat. no. 10494-1-AP; Wuhan Sanying Biotechnology).

Techniques: Incubation, Western Blot, In Situ

Journal: bioRxiv

Article Title: Hypoxia induced oxidative stress and endoplasmic reticulum stress promoted myocardial cell fibrosis

doi: 10.1101/2023.06.24.546381

Figure Lengend Snippet: Hypoxia induce ER stress in HL-1 cells. Expression of GRP78 after different intermittent hypoxia times in HL-1 cells (a, b). Expression of CHOP after different intermittent hypoxia times in HL-1 cells (c, d). Data shown are represented as the means ± S.E.M. from three se parate experiments. **P<0.01 vs. control, ***P<0.001 vs. control.

Article Snippet: mouse monoclonal antibodies against collagen I (Sigma; C2456) or α-smooth muscle actin (αSMA, sigma; A2547) and Rabbit monoclonal antibodies against glucose regulated protein 78 (GRP78, ABclonal; A4908) or CHOP (ABclonal; A20987) were used for western blot analysis.

Techniques: Expressing

Journal: bioRxiv

Article Title: Hypoxia induced oxidative stress and endoplasmic reticulum stress promoted myocardial cell fibrosis

doi: 10.1101/2023.06.24.546381

Figure Lengend Snippet: Antioxidant drugs NAC reduce cellular levels of ROS, and reduce GRP78 and CHOP expression in HL-1 cells. ROS levels in hypoxia-induced HL-1 cells were measured after 5mM NAC treatment (a, b). Effect of 5mM NAC on GRP78 and CHOP expression in HL-1 cells (c). Data shown are represented as the means ± S.E.M. from three separate experiments. *P<0.05 vs. control, **P<0.01 vs. control.

Article Snippet: mouse monoclonal antibodies against collagen I (Sigma; C2456) or α-smooth muscle actin (αSMA, sigma; A2547) and Rabbit monoclonal antibodies against glucose regulated protein 78 (GRP78, ABclonal; A4908) or CHOP (ABclonal; A20987) were used for western blot analysis.

Techniques: Expressing