Journal: Cell reports

Article Title: Xbp1-independent Ire1 signaling is required for photoreceptor differentiation and rhabdomere morphogenesis in Drosophila

doi: 10.1016/j.celrep.2013.09.046

Figure Lengend Snippet: Ire1 is required for Rh1 delivery into the rhabdomere. (A) 72 h pupal eye with eyeless-Flipase induced clones of PBac{WH}Ire1f02170 homozygous cells, labeled by the absence of myrGFP (green), show defective delivery of Rh1 (blue) into the rhabdomeres (actin, red). The magnified mutant and control ommatidia are indicated with white squares. (B) Rh1 (blue) delivery into the rhabdomeres is normal in clones of a precise excision of PBac{WH}Ire1f02170 (labeled by the absence of myrGFP, green). (C) GMR-Gal4>UAS-Ire1 expression rescues Rh1 (blue) delivery into the rhabdomeres of PBac{WH}Ire1f02170 homozygous cells (labeled by the absence of DsRed). (D) The CH322-07H05 genomic construct rescues Rh1 (blue) delivery into the rhabdomeres of PBac{WH}Ire1f02170 homozygous cells (absence of myrGFP, green). (E–G) PBac{WH}Ire1f02170 homozygous cells (absence of myrGFP, green) have reduced levels (arrows) of (E) BiP/GRP78 (blue and monochrome) and (F) ninaA (blue and monochrome), but not (G) Cnx (blue and monochrome). (H) Western blot analysis of Rh1 mature (34 kDa) and immature (40 kDa) forms from adult head protein extracts of yw (WT, positive control), FRT82BPBac{WH}Ire1f02170/FRT82BCL,GMR-hid (whole eye is composed of homozygous Ire1 mutant cells as in (Stowers and Schwarz, 1999)), ninaE17 (Rh1 protein null) and 2 ninaA mutations (ninaA1=ninaAP228 and ninaAE110V). Tubulin is used as loading control. Scale bars, 10 μm.

Article Snippet: Primary antibodies were as follows: rat anti-ELAV (7E8A10, DSHB), guinea pig anti-homothorax ( Wildonger et al., 2005 ), guinea pig anti-Runt ( Duffy et al., 1991 ), rat anti-cadherin (DCAD2, DSHB), mouse anti-armadillo (N2 7A1, DSHB), rabbit anti-aPKC (Santa Cruz, sc-216), mouse anti-Crumbs (Cq4, DSHB), mouse anti-spam (21A6, DSHB), mouse anti-Rh1 (4C5, DSHB), mouse anti-tubulin (12G10, DSHB), guinea pig anti-Bip/GRP78 (Hsc-3) ( Ryoo et al., 2007 ), rabbit anti-Bip/GRP78 (StressMarq Biosciences, SPC-180), guinea pig anti-ATF4 ( Kang et al., 2012 ), rabbit anti-calnexin ( Rosenbaum et al., 2006 ), rabbit anti-ninaA ( Baker et al., 1994 ), rabbit anti-Pdi (Abcam, ab2792), rabbit anti-fatp ( Dourlen et al., 2012 ) and mouse anti-Sparc ( Portela et al., 2010 ).

Techniques: Clone Assay, Labeling, Mutagenesis, Expressing, Construct, Western Blot, Positive Control

Journal: Cell reports

Article Title: Xbp1-independent Ire1 signaling is required for photoreceptor differentiation and rhabdomere morphogenesis in Drosophila

doi: 10.1016/j.celrep.2013.09.046

Figure Lengend Snippet: Xbp1 is not required for Spacemaker/Eyes shut secretion or Rh1 delivery into the rhabdomere. (A) Schematic of Xbp1 genomic region with the localization of P[lacW]Xbp1K13803, P[PTT-GB]Xbp1CB02061and P[SUPor-P]CG9418KG05183. Excisions 81 and 79 delete the DNA binding domain of Xbp1 and were generated from jumps of P[PTT-GB]Xbp1CB02061. In excision 81, the sequence from 4bp to 622bp downstream from Xbp1 start codon (ATG) is deleted. In excision 79, the sequence between 260bp upstream and 748bp downstream from Xbp1 start codon (ATG) is deleted. In excision 250, the open reading frame of Xbp1 is deleted from 5bp after the start codon, together with CG9418 and CG15657, until 2R:17036107, in the inter-genic region between CG15657 and CG30389. Excision 250 was generated from a jump of P[SUPor-P]CG9418KG05183. (B) By 62 h pupation, eyeless-Flipase induced clones of cells homozygous for Excision 79, labeled by the absence of myrGFP (green), have normal levels of Spam/Eys (blue) in the IRS. The rhabdomeres are stained with actin (red). (C) At 72 h. pupa, Rh1 (blue) is delivered to the rhabdomere in photoreceptors homozygous for Excision 79, labeled by the absence of myrRFP (red), but Xbp1 mutant photoreceptors have lower levels of Rh1 in the rhabdomere in than wild type (monochrome magnifications with wild type and mutant ommatidia). (D) At 82 h. pupa, photoreceptors homozygous for Excision 79 present higher levels of Rh1 in the rhabdomere than wild-type. Excision 79 homozygous photoreceptors show even distribution of Rh1 in the rhabdomere, while wild-type show the typical crescent-shaped gradient of concentration with higher levels at the rhabdomere base (monochrome magnifications). (E) Quantification of Rh1 intensity. Average of the ratio of Rh1 fluorescence intensity divided by respective area between pairs of adjacent Excision 79 homozygous and wild-type ommatidia, at 72 and 82 hours of pupal development (FluoEx79/AreaEx79/FluoWT/AreaWT). Around 70 pairs of Excision 79 homozygous and wild-type ommatidia were quantified at each time point. At 72 h. pupa, Excision 79 homozygous photoreceptors have reduced levels of (F) BiP/GRP78 (blue and monochrome) and (G) ninaA (blue and monochrome) but not (H) Cnx (blue and monochrome). Scale bars, 10 μm.

Article Snippet: Primary antibodies were as follows: rat anti-ELAV (7E8A10, DSHB), guinea pig anti-homothorax ( Wildonger et al., 2005 ), guinea pig anti-Runt ( Duffy et al., 1991 ), rat anti-cadherin (DCAD2, DSHB), mouse anti-armadillo (N2 7A1, DSHB), rabbit anti-aPKC (Santa Cruz, sc-216), mouse anti-Crumbs (Cq4, DSHB), mouse anti-spam (21A6, DSHB), mouse anti-Rh1 (4C5, DSHB), mouse anti-tubulin (12G10, DSHB), guinea pig anti-Bip/GRP78 (Hsc-3) ( Ryoo et al., 2007 ), rabbit anti-Bip/GRP78 (StressMarq Biosciences, SPC-180), guinea pig anti-ATF4 ( Kang et al., 2012 ), rabbit anti-calnexin ( Rosenbaum et al., 2006 ), rabbit anti-ninaA ( Baker et al., 1994 ), rabbit anti-Pdi (Abcam, ab2792), rabbit anti-fatp ( Dourlen et al., 2012 ) and mouse anti-Sparc ( Portela et al., 2010 ).

Techniques: Binding Assay, Generated, Sequencing, Clone Assay, Labeling, Staining, Mutagenesis, Concentration Assay, Fluorescence

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: Scientific Reports

Article Title: Neural tube opening and abnormal extraembryonic membrane development in SEC23A deficient mice

doi: 10.1038/srep15471

Figure Lengend Snippet: ( A ) Real-time RT-PCR quantification of select UPR genes in E9.5–E11.5 yolk sac and E11.5 amnion. Expression of the GAPDH gene was used as a reference. Data are mean ± SD. Asterisks indicate statistically significant difference between WT and Sec23a gt/gt samples (* P < 0.05, ** P < 0.01). ( B ) TUNEL staining (green) was performed for E11.5 amnion. Nuclei were visualized by DAPI staining (blue). TUNEL-positive cells were quantified as percentages of total cell numbers. ( C ) Activation of the PERK pathway in Sec23a gt/gt yolk sacs. Equal amounts of extracts from E11.5 yolk sac were immunoblotted for eIF2α, the phospohorylated eIF2α (p-eIF2α), GRP78 and β-actin.

Article Snippet: Other antibodies used include: mouse monoclonal anti-KDEL (MBL; 1:100), mouse monoclonal anti-PDI (Assay Designs; 1:200), rabbit polyclonal anti-Ki67 (Abcam; 1:500), rabbit polyclonal anti-fibronectin (Abcam; 1:100), mouse monoclonal anti-eIF2α and rabbit polyclonal anti-p-eIF2α (Cell Signaling; 1:1000), mouse monoclonal anti-GRP78 (MBL; 1:1000), mouse monoclonal anti-GAPDH (Sigma; 1:1000) and β-actin (Sigma; 1:10000).

Techniques: Quantitative RT-PCR, Expressing, TUNEL Assay, Staining, Activation Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Rosuvastatin protects against endothelial cell apoptosis in vitro and alleviates atherosclerosis in ApoE -/- mice by suppressing endoplasmic reticulum stress

doi: 10.3892/etm.2020.8733

Figure Lengend Snippet: Effect of rosuvastatin on the expression of GRP78 and p-IRE1α in HUVECs induced by ox-LDL. (A) The expression of GRP78 and p-IRE1α in HUVECs. (B and C) The densitometric analysis of GRP78 and p-IRE1α, the phosphorylation of IRE1α expressed as the ratio of p-IRE1α to total IRE1α. GAPDH was the loading control and the subjacent band is the target band of GRP78. The densitometric analysis values expressed as the mean ± standard deviation. ** P<0.01 vs. control group; # P<0.05 vs. ox-LDL; ## P<0.01 vs. ox-LDL group. GRP78, glucose-regulated protein 78; p-, phosphorylated; IRE1α, p-inositol-requiring protein 1α; HUVECs, human umbilical vascular endothelial cells.

Article Snippet: TRIzol ® reagent was from Thermo Fisher Scientific, Inc. TransScript Green Two-Step RT-qPCR SuperMix (cat. no. AQ201-01) was from Beijing Transgen Biotech Co., Ltd. Caspase-12 fluorometric assay kit was from BioVision, Inc. An anti-eIF2α polyclonal antibody (cat. no. BS3651), an anti-GRP78 polyclonal antibody (cat. no. BS1154) and an anti-PERK polyclonal antibody (cat. no. BS2156) were purchased from Bioworld Technology, Inc. An anti-phosphorylated (p-)eIF2α (Ser 51) monoclonal antibody (3398) and an anti-IRE1α monoclonal antibody (cat. no. 3294) were from Cell Signaling Technology, Inc. Anti-p-PERK (cat. no. 40294) polyclonal antibody and p-IRE1α (cat. no. 16927) polyclonal antibody were obtained from Thermo Fisher Scientific, Inc.

Techniques: Expressing, Standard Deviation

Journal: Experimental and Therapeutic Medicine

Article Title: Rosuvastatin protects against endothelial cell apoptosis in vitro and alleviates atherosclerosis in ApoE -/- mice by suppressing endoplasmic reticulum stress

doi: 10.3892/etm.2020.8733

Figure Lengend Snippet: Effect of rosuvastatin on ER stress in aortic intima of atherosclerotic mice. Representative immunohistochemical staining and content quantification of GRP78, p-PERK, p-IRE1α and p-elF2α in aortic intima and plaque, magnification, x 400, scale bar = 50 µm. The density means expressed as the mean ± standard deviation. ** P<0.01 vs. Control group; # P<0.01 vs. Model group. ER GRP78, glucose-regulated protein 78; p-, phosphorylated; PERK, protein kinase RNA-like ER kinase; IRE1α, p-inositol-requiring protein 1α; eIF2α, inositol-requiring protein 1α.

Article Snippet: TRIzol ® reagent was from Thermo Fisher Scientific, Inc. TransScript Green Two-Step RT-qPCR SuperMix (cat. no. AQ201-01) was from Beijing Transgen Biotech Co., Ltd. Caspase-12 fluorometric assay kit was from BioVision, Inc. An anti-eIF2α polyclonal antibody (cat. no. BS3651), an anti-GRP78 polyclonal antibody (cat. no. BS1154) and an anti-PERK polyclonal antibody (cat. no. BS2156) were purchased from Bioworld Technology, Inc. An anti-phosphorylated (p-)eIF2α (Ser 51) monoclonal antibody (3398) and an anti-IRE1α monoclonal antibody (cat. no. 3294) were from Cell Signaling Technology, Inc. Anti-p-PERK (cat. no. 40294) polyclonal antibody and p-IRE1α (cat. no. 16927) polyclonal antibody were obtained from Thermo Fisher Scientific, Inc.

Techniques: Immunohistochemical staining, Staining, Standard Deviation