Journal: Journal of Cellular and Molecular Medicine

Article Title: Reticulocalbin 1 is required for proliferation and migration of non‐small cell lung cancer cells regulated by osteoblast‐conditioned medium

doi: 10.1111/jcmm.17040

Figure Lengend Snippet: Information of antibodies

Article Snippet: p‐PERK(phosphoT980) , Bioss Bs−3330R , 1:1000 , 4℃ overnight.

Techniques:

Journal: Neurobiology of disease

Article Title: Calcineurin β protects brain after injury by activating the unfolded protein response

doi: 10.1016/j.nbd.2016.06.011

Figure Lengend Snippet: CNβ physically interacts with PERK and promotes PERK phosphorylation and oligomerization. (A) C8D1A type I astrocytes were treated with vehicle (DMSO; Con) or 1 µM of thapsigargin (Tg) for 1 h. Cell lysates were analyzed for total (T) and phosphorylated (P) PERK and eIF2α using immunoblots. (B) Densitometry histograms after normalization to T-PERK or T-eIF2α, respectively (n = 3, mean ± SEM, ** p < 0.01 and *** p < 0.001 by unpaired two-tailed Student’s t -test). (C) Primary mouse astrocytes were treated as indicated in (A) and then cross-linked with disuccinimidyl suberate (DSS) for 30 min. Immunoprecipitation (IP) was performed with anti-CNβ antibody and subsequent blots were probed with anti-PERK antibody. (D) Quantification of P-PERK/T-PERK densitometric ratio in (C) (n = 3, mean ± SEM, * p < 0.05 by unpaired two-tailed Student’s t test). (E) GST pull-down assay with either 8 nM of CNα or CNβ. The proteins were incubated with glutathione sepharose 4B for 1 h, resolved on a 12% SDS-polyacrylamide gel and probed with an anti-calcineurin PAN-A antibody. CN pull-down levels are shown for GST alone and GST-cPERK. (F) Densitometric histogram of (E) (n = 3, mean ± SEM, ** p < 0.01 by unpaired two-tailed Student’s t test). (G) GST-cPERK was added to all reaction mixtures along with [g 32 P] ATP in the absence or the presence of either of 0.043 mM of CNα or CNβ. Reaction mixtures were run on SDS-PAGE and visualized by autoradiography. (H) Quantification of cPERK auto-phosphorylation density (ne = 5, mean ± SEM, * p < 0.05 by one-way ANOVA). (I) Recombinant His-CNβ and GST-cPERK were incubated at the concentrations indicated, in the presence of 0.3 mM of DSS cross-linker for 30 min at room temperature. The ensuing protein complexes were run on SDS-PAGE and detected by immunoblotting using an anti-PERK antibody. (J) Recombinant His-CNβ (1.2 mM) and GST-cPERK (0.01 mM) were incubated in the presence of 0.3 mM of DSS for 30 min at room temperature. The protein complexes were run on SDS-PAGE and detected by immunoblotting using antibodies against CNβ and PERK.

Article Snippet: Primary antibodies used were rabbit anti-phosphorylated PERK (conjugated to Alexa 647, #bs-3330R–A647; Bioss), rabbit anti-CNα (#13422-1-AP; Proteintech), rabbit anti-CNβ (#07–1439; Millipore), rabbit anti-phosphorylated eIF2α (P-eIF2α, #µA5 – 15133; Thermo scientific) and mouse anti-GFAP (#MAB 360; Millipore).

Techniques: Western Blot, Two Tailed Test, Immunoprecipitation, Pull Down Assay, Incubation, SDS Page, Autoradiography, Recombinant

Journal: International Journal of Molecular Medicine

Article Title: Human ether-à-go-go-related gene mutation L539fs/47-hERG leads to cell apoptosis through the endoplasmic reticulum stress pathway

doi: 10.3892/ijmm.2019.4049

Figure Lengend Snippet: Western blot analysis results for ERS-associated proteins. (A) The representative blotting images and (B) quantified data demonstrate elevated p-PERK, eIF2a, CHOP, Bax/Bcl-2, Caspase-12 and cleaved Caspase-3 level in L539fs/47-hERG cells, which can be reversed by 4-PBA. Each experiment was repeated at least three times. # P<0.05 between the three model cell groups; * P<0.05 between two subgroups treated with or without 4-PBA intervention. P<0.05 was considered to indicate a statistically significant difference. ERS, endoplasmic reticulum stress; hERG, human ether-à-go-go-related gene; WT, wild-type; 4PBA, 4-phenyl butyric acid; p-PERK, phosphorylated protein kinase R-like endoplasmic reticulum kinase; eIF2a; eukaryotic translation-initiation factor-2α; CHOP, C/EBP homologous protein; Bcl2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.

Article Snippet: The primary antibodies were as follows: hERG (sc-377388; 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), GRP78 (cat. no. WL0781; 1:1,000; Wanleibio Co., Ltd., Shanghai, China), phosphorylated (p-)PERK (cat. no. bs-23340R; 1:500; Bioss, Beijing, China), p-eIF2α (cat. no. 3398; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), CHOP (cat. no. WL00880; 1:1,000), Bax (cat. no. WL01637; 1:500), Bcl-2 (cat. no. WL01556; 1:500), cleaved-caspase-3 (cat. no. WL01857; 1:500; all Wanleibio Co., Ltd.), caspase-12 (cat. no. 2202; 1:1,000) and GAPDH (cat. no. sc-25778; 1:1,000; both Santa Cruz Biotechnology, Inc.).

Techniques: Western Blot

Journal: International Journal of Molecular Medicine

Article Title: Human ether-à-go-go-related gene mutation L539fs/47-hERG leads to cell apoptosis through the endoplasmic reticulum stress pathway

doi: 10.3892/ijmm.2019.4049

Figure Lengend Snippet: Proposed mechanisms of mutated hERG-induced cell apoptosis. Mutated L539fs/47-hERG protein retained in the ER lumen provokes ERS. Elevated expression levels of GRP78 activate the PERK-eIF2α-CHOP pathway, which is important in the mechanism of L539fs/47-hERG-induced ERS-mediated cell apoptosis. ER-specific caspase-12 is involved in the apoptotic mechanisms caused by the hERG mutation through cleaving and activating caspase-3. The pair of apoptosis factors Bax/Bcl-2 also regulate L539fs/47-hERG-induced cell apoptosis. hERG, human ether-à-go-go-related gene; WT, wild-type; 4PBA, 4-phenyl butyric acid; ER, endoplasmic reticulum; ERS, ER stress; ATF6, activating transcription factor 6; IRE1, inositol-requiring enzyme 1; p-PERK, phosphorylated protein kinase R-like endoplasmic reticulum kinase; eIF2a; eukaryotic translation-initiation factor-2α; CHOP, C/EBP homologous protein; Bcl2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.

Article Snippet: The primary antibodies were as follows: hERG (sc-377388; 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), GRP78 (cat. no. WL0781; 1:1,000; Wanleibio Co., Ltd., Shanghai, China), phosphorylated (p-)PERK (cat. no. bs-23340R; 1:500; Bioss, Beijing, China), p-eIF2α (cat. no. 3398; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), CHOP (cat. no. WL00880; 1:1,000), Bax (cat. no. WL01637; 1:500), Bcl-2 (cat. no. WL01556; 1:500), cleaved-caspase-3 (cat. no. WL01857; 1:500; all Wanleibio Co., Ltd.), caspase-12 (cat. no. 2202; 1:1,000) and GAPDH (cat. no. sc-25778; 1:1,000; both Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Mutagenesis

Journal: International journal of molecular medicine

Article Title: FGF21 attenuates hypoxia‑induced dysfunction and apoptosis in HPAECs through alleviating endoplasmic reticulum stress.

doi: 10.3892/ijmm.2018.3705

Figure Lengend Snippet: Figure 4. FGF21 alleviates the hypoxia‑induced ERS by modulating the expression of ERS‑related proteins in HPAECs. HPAECs were treated as indicated and protein expression levels were examined by western blotting. Representative blots and quantification are shown for (A) BiP, (B) p‑PERK and PERK, (C) the ratio of p‑PERK/PERK, (D) CHOP, (E) Bcl‑2. (F) Relative expression of the ERS‑dependent apoptotic protein caspase‑4 in HPAECs was analysed by western blotting. GAPDH was used as an internal control. All experiments were performed in triplicate, and data are expressed as the mean ± standard deviation (n=3). **P<0.01 vs. N group; ##P<0.01 vs. the H group; &&P<0.01 vs. the H+T group. FGF21, fibroblast growth factor 21; ERS, endoplasmic reticulum stress; HPAECs, human pulmonary arterial endothelial cells; BiP, binding immunoglobulin protein; p‑, phosphorylated; PERK, protein kinase R‑like endoplasmic reticulum kinase; CHOP, transcrip- tion factor C/EBP homologous protein; Bcl‑2, B cell lymphoma-2; N, normoxia; H, hypoxia; F, FGF21; S, salubrinal; T, tunicamycin.

Article Snippet: Rabbit antibodies against phosphorylated (p-) PERK were purchased from Biorbyt (cambridge, UK).

Techniques: Expressing, Western Blot, Control, Standard Deviation, Binding Assay

Journal: International journal of molecular medicine

Article Title: FGF21 attenuates hypoxia‑induced dysfunction and apoptosis in HPAECs through alleviating endoplasmic reticulum stress.

doi: 10.3892/ijmm.2018.3705

Figure Lengend Snippet: Figure 6. Effect of FGF21 on NO and ET‑1 secretion in HPAECs. (A) HPAECs were treated as indicated and at the end of treatment, the cell culture medium was collected and assayed by ELISA for the levels of secreted NO and (B) the levels of secreted ET‑1. Data are expressed as the mean ± standard deviation (n=3). **P<0.01 vs. the N group; #P<0.05 and ##P<0.01 vs. the H group; &&P<0.01 vs. the H+T group. (C) Schematic of the proposed mechanism by which FGF21 attenuates hypoxia‑induced apoptosis and dysfunction by alleviating ERS in HPAECs. FGF21, fibroblast growth factor 21; NO, nitric oxide; ET‑1, endothelin‑1; HPAECs, human pulmonary arterial endothelial cells; ERS, endoplasmic reticulum stress; N, normoxia; H, hypoxia; F, FGF21; S, salubrinal; T, tunicamycin; BiP, binding immunoglobulin protein; p‑, phosphorylated; PERK, protein kinase R‑like endoplasmic reticulum kinase; CHOP, transcription factor C/EBP homologous protein; Bcl‑2, B cell lymphoma-2.

Article Snippet: Rabbit antibodies against phosphorylated (p-) PERK were purchased from Biorbyt (cambridge, UK).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation, Binding Assay