Journal: Genes

Article Title: STIM1–Orai1 Interaction Exacerbates LPS-Induced Inflammation and Endoplasmic Reticulum Stress in Bovine Hepatocytes through Store-Operated Calcium Entry

doi: 10.3390/genes13050874

Figure Lengend Snippet: The list of primer sequences for RT-qPCR.

Article Snippet: The membrane was blocked with 7% skim milk or BSA (for phosphorylated proteins) at 25 °C for 2 h. The samples were subsequently incubated at 4 °C overnight with protein-specific antibodies (all antibodies were diluted with TBST 1:1000): NF-κB p65 (AF0246, Beyotime), phosphorus-NF-κB P65 (AF5881, Beyotime), IκB α (AF2176, Beyotime), phosphoric acid IκBα (AF1870, Beyotime), TNF α (A11534, Abclonal, Wuhan, China), STIM1 (AF2614, Beyotime), Orai1 (66223-1-Ig, ProteinTech, Thermo Fisher Scientific, Waltham, MA, USA), GRP78 (AF0171, Beyotime), CHOP (AC532, Beyotime), phospho-PERK (AF5902; Cell Signailing, Boston, MA, USA), PERK (D11A8, Cell Signaling), IRE1 α (AI601, Beyotime), phospho-IRE1α (AF5842, Beyotime), and ATF6 (AF6243, Beyotime) at 4 °C overnight.

Techniques: Sequencing

Journal: Genes

Article Title: STIM1–Orai1 Interaction Exacerbates LPS-Induced Inflammation and Endoplasmic Reticulum Stress in Bovine Hepatocytes through Store-Operated Calcium Entry

doi: 10.3390/genes13050874

Figure Lengend Snippet: Effects of different concentrations of LPS on bovine hepatocytes (BHEC). When the adherent density of BHEC reached about 70%, the BHEC was treated with LPS at 0, 4, 8, 12, 16, or 20 μg/mL for 12 h. Gene expression was normalized with GAPDH as the reference protein. ( A ) Gene expression of STIM1 and Orai1 in BHECs. ( B , C ) Gene expression levels of NF-κB, IκB, IL-6, IL-8, and TNF-α. ( D , E ) Changes in endoplasmic reticulum stress (ERS)-related genes (PERK, IRE1, ATF6, GRP78, CHOP) under different doses of LPS. Error bars represent the means ± SEM ( n = 3, n = 3). * p < 0.05, ** p < 0.01 compared with the controls. ( F ) After treatment with 16 μg/mL LPS for 1 h, the fluorescence signals and position changes of STIM1, Orai1, and p65 in BHEC were observed by confocal immunofluorescence microscopy. DAPI blue fluorescence was used to label the location of the nucleus; FITC green fluorescence was used to label the target proteins.

Article Snippet: The membrane was blocked with 7% skim milk or BSA (for phosphorylated proteins) at 25 °C for 2 h. The samples were subsequently incubated at 4 °C overnight with protein-specific antibodies (all antibodies were diluted with TBST 1:1000): NF-κB p65 (AF0246, Beyotime), phosphorus-NF-κB P65 (AF5881, Beyotime), IκB α (AF2176, Beyotime), phosphoric acid IκBα (AF1870, Beyotime), TNF α (A11534, Abclonal, Wuhan, China), STIM1 (AF2614, Beyotime), Orai1 (66223-1-Ig, ProteinTech, Thermo Fisher Scientific, Waltham, MA, USA), GRP78 (AF0171, Beyotime), CHOP (AC532, Beyotime), phospho-PERK (AF5902; Cell Signailing, Boston, MA, USA), PERK (D11A8, Cell Signaling), IRE1 α (AI601, Beyotime), phospho-IRE1α (AF5842, Beyotime), and ATF6 (AF6243, Beyotime) at 4 °C overnight.

Techniques: Expressing, Fluorescence, Immunofluorescence, Microscopy

Journal: Genes

Article Title: STIM1–Orai1 Interaction Exacerbates LPS-Induced Inflammation and Endoplasmic Reticulum Stress in Bovine Hepatocytes through Store-Operated Calcium Entry

doi: 10.3390/genes13050874

Figure Lengend Snippet: Effects of the overexpression of STIM1 on inflammation and endoplasmic reticulum stress (ERS) in BHEC. Treatments: BHEC were transfected with overexpressed plasmids and cultured for 48 h. CON, complete control groups; NC, transfected blank plasmid and cultured for 48 h; OE-STIM1, BHEC transfected with overexpressed STIM1 plasmid cultured for 48 h. All groups were cultured without LPS. (A,B) Expression of ( A ) STIM1 and ( B ) Orai1 in BHECs. ( C , D ) Expression of ( C ) IL-6, IL-8, and TNF-α, and ( D ) NF-κB and IκB associated with the inflammatory response. ( E ) Expression levels of ERS-related genes (PERK, IRE1, ATF6, GRP78, and CHOP) in BHEC. Gene expression was normalized to the expression of GAPDH. The results are presented as the means ± SEM., ** p < 0.01.

Article Snippet: The membrane was blocked with 7% skim milk or BSA (for phosphorylated proteins) at 25 °C for 2 h. The samples were subsequently incubated at 4 °C overnight with protein-specific antibodies (all antibodies were diluted with TBST 1:1000): NF-κB p65 (AF0246, Beyotime), phosphorus-NF-κB P65 (AF5881, Beyotime), IκB α (AF2176, Beyotime), phosphoric acid IκBα (AF1870, Beyotime), TNF α (A11534, Abclonal, Wuhan, China), STIM1 (AF2614, Beyotime), Orai1 (66223-1-Ig, ProteinTech, Thermo Fisher Scientific, Waltham, MA, USA), GRP78 (AF0171, Beyotime), CHOP (AC532, Beyotime), phospho-PERK (AF5902; Cell Signailing, Boston, MA, USA), PERK (D11A8, Cell Signaling), IRE1 α (AI601, Beyotime), phospho-IRE1α (AF5842, Beyotime), and ATF6 (AF6243, Beyotime) at 4 °C overnight.

Techniques: Over Expression, Transfection, Cell Culture, Plasmid Preparation, Expressing

Journal: Genes

Article Title: STIM1–Orai1 Interaction Exacerbates LPS-Induced Inflammation and Endoplasmic Reticulum Stress in Bovine Hepatocytes through Store-Operated Calcium Entry

doi: 10.3390/genes13050874

Figure Lengend Snippet: Effects of STIM1 overexpression on LPS-induced inflammation and endoplasmic reticulum stress (ERS) in BHEC. Treatments: BHEC were transfected with overexpressed plasmids and cultured for 48 h. NC, transfected blank plasmid cultured for 48 h without LPS treatment; OE-STIM1, BHEC transfected with overexpressed STIM1 plasmids that were cultured for 48 h without LPS treatment; LPS, transfected blank plasmids cultured for 48 h, then treated with 16 μg/mL LPS for 1 h; OE-STIM1 + LPS, BHEC transfected with overexpressed STIM1 plasmids that were cultured for 48 h and then treated with 16 μg/mL LPS for 1 h. Gene expression of STIM1/Orai1 ( A ), inflammatory cytokines (NF-κB, IκB, IL-6, IL-8, and TNF-α) ( B ), and ERS-related genes (PERK, IRE1, ATF6, GRP78, and CHOP) ( C ) in BHEC. The protein abundance of STIM1/Orai1 ( D ), inflammatory cytokines (PKCα, p65, p-p65, IκB, p-IκB, and TNFα) ( E ), and ERS-related genes (PERK, IRE1, ATF6, GRP78, and CHOP) ( F ) was normalized to the abundance of β-actin. The results are presented as the means ± SEM. * p < 0.05, ** p < 0.01 indicate that the LPS groups and the OE-STIM1 groups were significantly different from control group. # p < 0.05, ## p < 0.01 indicates the significant difference between the OE-STIM1 + LPS groups and the NC + LPS groups; n.s. indicates no difference between the two groups.

Article Snippet: The membrane was blocked with 7% skim milk or BSA (for phosphorylated proteins) at 25 °C for 2 h. The samples were subsequently incubated at 4 °C overnight with protein-specific antibodies (all antibodies were diluted with TBST 1:1000): NF-κB p65 (AF0246, Beyotime), phosphorus-NF-κB P65 (AF5881, Beyotime), IκB α (AF2176, Beyotime), phosphoric acid IκBα (AF1870, Beyotime), TNF α (A11534, Abclonal, Wuhan, China), STIM1 (AF2614, Beyotime), Orai1 (66223-1-Ig, ProteinTech, Thermo Fisher Scientific, Waltham, MA, USA), GRP78 (AF0171, Beyotime), CHOP (AC532, Beyotime), phospho-PERK (AF5902; Cell Signailing, Boston, MA, USA), PERK (D11A8, Cell Signaling), IRE1 α (AI601, Beyotime), phospho-IRE1α (AF5842, Beyotime), and ATF6 (AF6243, Beyotime) at 4 °C overnight.

Techniques: Over Expression, Transfection, Cell Culture, Plasmid Preparation, Expressing

Journal: Genes

Article Title: STIM1–Orai1 Interaction Exacerbates LPS-Induced Inflammation and Endoplasmic Reticulum Stress in Bovine Hepatocytes through Store-Operated Calcium Entry

doi: 10.3390/genes13050874

Figure Lengend Snippet: Schematic representation shows that Toll-like receptors are stimulated by LPS, activate the STIM1/Orai1-mediated SOCE channel, cause endoplasmic reticulum stress (ERS), and turn on the NF-κB signaling pathway to induce inflammation. The upregulation of STIM1 intensifies LPS-induced inflammation and endoplasmic reticulum stress (ERS), whereas the inhibition of Orai1 alleviates the inflammatory effects of LPS and ERS. LPS, lipopolysaccharide; STIM1, stromal interaction molecule 1; Orai1, calcium release-activated calcium channel protein 1; NF-κB, nuclear factor-kappa B; IκB, inhibitor of NF-κB; PERK, pancreatic elf-2 kinase pancreatic ER kinase; ATF6, activating transcription factor 6; IRE1, inositol requiring enzyme 1; GRP78, glucose-regulated protein 78; CHOP, C/EBP homologous protein; IL-6, interleukin-6; IL-8, interleukin-8; TNF-α, tumor necrosis factor-α.

Article Snippet: The membrane was blocked with 7% skim milk or BSA (for phosphorylated proteins) at 25 °C for 2 h. The samples were subsequently incubated at 4 °C overnight with protein-specific antibodies (all antibodies were diluted with TBST 1:1000): NF-κB p65 (AF0246, Beyotime), phosphorus-NF-κB P65 (AF5881, Beyotime), IκB α (AF2176, Beyotime), phosphoric acid IκBα (AF1870, Beyotime), TNF α (A11534, Abclonal, Wuhan, China), STIM1 (AF2614, Beyotime), Orai1 (66223-1-Ig, ProteinTech, Thermo Fisher Scientific, Waltham, MA, USA), GRP78 (AF0171, Beyotime), CHOP (AC532, Beyotime), phospho-PERK (AF5902; Cell Signailing, Boston, MA, USA), PERK (D11A8, Cell Signaling), IRE1 α (AI601, Beyotime), phospho-IRE1α (AF5842, Beyotime), and ATF6 (AF6243, Beyotime) at 4 °C overnight.

Techniques: Inhibition

Journal: Genes

Article Title: STIM1–Orai1 Interaction Exacerbates LPS-Induced Inflammation and Endoplasmic Reticulum Stress in Bovine Hepatocytes through Store-Operated Calcium Entry

doi: 10.3390/genes13050874

Figure Lengend Snippet: The list of primer sequences for RT-qPCR.

Article Snippet: The membrane was blocked with 7% skim milk or BSA (for phosphorylated proteins) at 25 °C for 2 h. The samples were subsequently incubated at 4 °C overnight with protein-specific antibodies (all antibodies were diluted with TBST 1:1000): NF-κB p65 (AF0246, Beyotime), phosphorus-NF-κB P65 (AF5881, Beyotime), IκB α (AF2176, Beyotime), phosphoric acid IκBα (AF1870, Beyotime), TNF α (A11534, Abclonal, Wuhan, China), STIM1 (AF2614, Beyotime), Orai1 (66223-1-Ig, ProteinTech, Thermo Fisher Scientific, Waltham, MA, USA), GRP78 (AF0171, Beyotime), CHOP (AC532, Beyotime), phospho-PERK (AF5902; Cell Signailing, Boston, MA, USA), PERK (D11A8, Cell Signaling), IRE1 α (AI601, Beyotime), phospho-IRE1α (AF5842, Beyotime), and ATF6 (AF6243, Beyotime) at 4 °C overnight.

Techniques: Sequencing

Journal: Genes

Article Title: STIM1–Orai1 Interaction Exacerbates LPS-Induced Inflammation and Endoplasmic Reticulum Stress in Bovine Hepatocytes through Store-Operated Calcium Entry

doi: 10.3390/genes13050874

Figure Lengend Snippet: Effects of different concentrations of LPS on bovine hepatocytes (BHEC). When the adherent density of BHEC reached about 70%, the BHEC was treated with LPS at 0, 4, 8, 12, 16, or 20 μg/mL for 12 h. Gene expression was normalized with GAPDH as the reference protein. ( A ) Gene expression of STIM1 and Orai1 in BHECs. ( B , C ) Gene expression levels of NF-κB, IκB, IL-6, IL-8, and TNF-α. ( D , E ) Changes in endoplasmic reticulum stress (ERS)-related genes (PERK, IRE1, ATF6, GRP78, CHOP) under different doses of LPS. Error bars represent the means ± SEM ( n = 3, n = 3). * p < 0.05, ** p < 0.01 compared with the controls. ( F ) After treatment with 16 μg/mL LPS for 1 h, the fluorescence signals and position changes of STIM1, Orai1, and p65 in BHEC were observed by confocal immunofluorescence microscopy. DAPI blue fluorescence was used to label the location of the nucleus; FITC green fluorescence was used to label the target proteins.

Article Snippet: The membrane was blocked with 7% skim milk or BSA (for phosphorylated proteins) at 25 °C for 2 h. The samples were subsequently incubated at 4 °C overnight with protein-specific antibodies (all antibodies were diluted with TBST 1:1000): NF-κB p65 (AF0246, Beyotime), phosphorus-NF-κB P65 (AF5881, Beyotime), IκB α (AF2176, Beyotime), phosphoric acid IκBα (AF1870, Beyotime), TNF α (A11534, Abclonal, Wuhan, China), STIM1 (AF2614, Beyotime), Orai1 (66223-1-Ig, ProteinTech, Thermo Fisher Scientific, Waltham, MA, USA), GRP78 (AF0171, Beyotime), CHOP (AC532, Beyotime), phospho-PERK (AF5902; Cell Signailing, Boston, MA, USA), PERK (D11A8, Cell Signaling), IRE1 α (AI601, Beyotime), phospho-IRE1α (AF5842, Beyotime), and ATF6 (AF6243, Beyotime) at 4 °C overnight.

Techniques: Gene Expression, Fluorescence, Immunofluorescence, Microscopy

Journal: Genes

Article Title: STIM1–Orai1 Interaction Exacerbates LPS-Induced Inflammation and Endoplasmic Reticulum Stress in Bovine Hepatocytes through Store-Operated Calcium Entry

doi: 10.3390/genes13050874

Figure Lengend Snippet: Effects of the overexpression of STIM1 on inflammation and endoplasmic reticulum stress (ERS) in BHEC. Treatments: BHEC were transfected with overexpressed plasmids and cultured for 48 h. CON, complete control groups; NC, transfected blank plasmid and cultured for 48 h; OE-STIM1, BHEC transfected with overexpressed STIM1 plasmid cultured for 48 h. All groups were cultured without LPS. (A,B) Expression of ( A ) STIM1 and ( B ) Orai1 in BHECs. ( C , D ) Expression of ( C ) IL-6, IL-8, and TNF-α, and ( D ) NF-κB and IκB associated with the inflammatory response. ( E ) Expression levels of ERS-related genes (PERK, IRE1, ATF6, GRP78, and CHOP) in BHEC. Gene expression was normalized to the expression of GAPDH. The results are presented as the means ± SEM., ** p < 0.01.

Article Snippet: The membrane was blocked with 7% skim milk or BSA (for phosphorylated proteins) at 25 °C for 2 h. The samples were subsequently incubated at 4 °C overnight with protein-specific antibodies (all antibodies were diluted with TBST 1:1000): NF-κB p65 (AF0246, Beyotime), phosphorus-NF-κB P65 (AF5881, Beyotime), IκB α (AF2176, Beyotime), phosphoric acid IκBα (AF1870, Beyotime), TNF α (A11534, Abclonal, Wuhan, China), STIM1 (AF2614, Beyotime), Orai1 (66223-1-Ig, ProteinTech, Thermo Fisher Scientific, Waltham, MA, USA), GRP78 (AF0171, Beyotime), CHOP (AC532, Beyotime), phospho-PERK (AF5902; Cell Signailing, Boston, MA, USA), PERK (D11A8, Cell Signaling), IRE1 α (AI601, Beyotime), phospho-IRE1α (AF5842, Beyotime), and ATF6 (AF6243, Beyotime) at 4 °C overnight.

Techniques: Over Expression, Transfection, Cell Culture, Control, Plasmid Preparation, Expressing, Gene Expression

Journal: Genes

Article Title: STIM1–Orai1 Interaction Exacerbates LPS-Induced Inflammation and Endoplasmic Reticulum Stress in Bovine Hepatocytes through Store-Operated Calcium Entry

doi: 10.3390/genes13050874

Figure Lengend Snippet: Effects of STIM1 overexpression on LPS-induced inflammation and endoplasmic reticulum stress (ERS) in BHEC. Treatments: BHEC were transfected with overexpressed plasmids and cultured for 48 h. NC, transfected blank plasmid cultured for 48 h without LPS treatment; OE-STIM1, BHEC transfected with overexpressed STIM1 plasmids that were cultured for 48 h without LPS treatment; LPS, transfected blank plasmids cultured for 48 h, then treated with 16 μg/mL LPS for 1 h; OE-STIM1 + LPS, BHEC transfected with overexpressed STIM1 plasmids that were cultured for 48 h and then treated with 16 μg/mL LPS for 1 h. Gene expression of STIM1/Orai1 ( A ), inflammatory cytokines (NF-κB, IκB, IL-6, IL-8, and TNF-α) ( B ), and ERS-related genes (PERK, IRE1, ATF6, GRP78, and CHOP) ( C ) in BHEC. The protein abundance of STIM1/Orai1 ( D ), inflammatory cytokines (PKCα, p65, p-p65, IκB, p-IκB, and TNFα) ( E ), and ERS-related genes (PERK, IRE1, ATF6, GRP78, and CHOP) ( F ) was normalized to the abundance of β-actin. The results are presented as the means ± SEM. * p < 0.05, ** p < 0.01 indicate that the LPS groups and the OE-STIM1 groups were significantly different from control group. # p < 0.05, ## p < 0.01 indicates the significant difference between the OE-STIM1 + LPS groups and the NC + LPS groups; n.s. indicates no difference between the two groups.

Article Snippet: The membrane was blocked with 7% skim milk or BSA (for phosphorylated proteins) at 25 °C for 2 h. The samples were subsequently incubated at 4 °C overnight with protein-specific antibodies (all antibodies were diluted with TBST 1:1000): NF-κB p65 (AF0246, Beyotime), phosphorus-NF-κB P65 (AF5881, Beyotime), IκB α (AF2176, Beyotime), phosphoric acid IκBα (AF1870, Beyotime), TNF α (A11534, Abclonal, Wuhan, China), STIM1 (AF2614, Beyotime), Orai1 (66223-1-Ig, ProteinTech, Thermo Fisher Scientific, Waltham, MA, USA), GRP78 (AF0171, Beyotime), CHOP (AC532, Beyotime), phospho-PERK (AF5902; Cell Signailing, Boston, MA, USA), PERK (D11A8, Cell Signaling), IRE1 α (AI601, Beyotime), phospho-IRE1α (AF5842, Beyotime), and ATF6 (AF6243, Beyotime) at 4 °C overnight.

Techniques: Over Expression, Transfection, Cell Culture, Plasmid Preparation, Gene Expression, Quantitative Proteomics, Control

Journal: Genes

Article Title: STIM1–Orai1 Interaction Exacerbates LPS-Induced Inflammation and Endoplasmic Reticulum Stress in Bovine Hepatocytes through Store-Operated Calcium Entry

doi: 10.3390/genes13050874

Figure Lengend Snippet: Protein expression and location of STIM1/Orai1 in BMEC. DAPI blue fluorescence was used to label the nuclear locations, FITC green fluorescence was used to label STIM1 protein, and Cy3 red fluorescence was used to label Orai1 protein. ImageJ was used to draw the line graph to show the fluorescence co-location trends.

Article Snippet: The membrane was blocked with 7% skim milk or BSA (for phosphorylated proteins) at 25 °C for 2 h. The samples were subsequently incubated at 4 °C overnight with protein-specific antibodies (all antibodies were diluted with TBST 1:1000): NF-κB p65 (AF0246, Beyotime), phosphorus-NF-κB P65 (AF5881, Beyotime), IκB α (AF2176, Beyotime), phosphoric acid IκBα (AF1870, Beyotime), TNF α (A11534, Abclonal, Wuhan, China), STIM1 (AF2614, Beyotime), Orai1 (66223-1-Ig, ProteinTech, Thermo Fisher Scientific, Waltham, MA, USA), GRP78 (AF0171, Beyotime), CHOP (AC532, Beyotime), phospho-PERK (AF5902; Cell Signailing, Boston, MA, USA), PERK (D11A8, Cell Signaling), IRE1 α (AI601, Beyotime), phospho-IRE1α (AF5842, Beyotime), and ATF6 (AF6243, Beyotime) at 4 °C overnight.

Techniques: Expressing, Fluorescence

Journal: Genes

Article Title: STIM1–Orai1 Interaction Exacerbates LPS-Induced Inflammation and Endoplasmic Reticulum Stress in Bovine Hepatocytes through Store-Operated Calcium Entry

doi: 10.3390/genes13050874

Figure Lengend Snippet: Effects of the Orai1 inhibitor BTP2 on LPS-induced inflammation and endoplasmic reticulum stress (ERS) in BHEC. Treatments: BHEC were cultured in a BTP2-free medium for 24 h. CON, complete control groups; LPS, cells treated with 16 μg/mL LPS for 1 h; BTP2, pretreatment with 8 μg/mL BTP2 for 6 h without LPS treatment; LPS + BTP2, 8 μg/mL BTP2 for 6 h, followed by 16 μg/mL LPS for 1 h. ( A – D ) Expression levels of STIM1/Orai1 and the genes related to inflammatory cytokines (NF-κB, IκB, IL-6, IL-8, and TNFα) and ERS (PERK, IRE1, ATF6, GRP78, and CHOP) were shown. Gene expression levels were normalized to that of GAPDH. ( E – G ) Protein abundance levels of STIM1/Orai1, PKCα, p65, p-p65, IκB, p-IκB, TNFα, PERK, p-PERK, IRE1, p-IRE1, ATF6, GRP78, and CHOP in BHEC, showing representative bands of the Western blot analysis and the quantified volume of specific bands. The results are presented as the means ± SEM. * p < 0.05, ** p < 0.01 indicate a significant difference between the LPS groups and the control groups; n.s. indicates that there was no significant difference between the BTP2 group and the CON group. # p < 0.05, ## p < 0.01 indicate a significant difference between the BTP2 pretreatment groups and the LPS groups.

Article Snippet: The membrane was blocked with 7% skim milk or BSA (for phosphorylated proteins) at 25 °C for 2 h. The samples were subsequently incubated at 4 °C overnight with protein-specific antibodies (all antibodies were diluted with TBST 1:1000): NF-κB p65 (AF0246, Beyotime), phosphorus-NF-κB P65 (AF5881, Beyotime), IκB α (AF2176, Beyotime), phosphoric acid IκBα (AF1870, Beyotime), TNF α (A11534, Abclonal, Wuhan, China), STIM1 (AF2614, Beyotime), Orai1 (66223-1-Ig, ProteinTech, Thermo Fisher Scientific, Waltham, MA, USA), GRP78 (AF0171, Beyotime), CHOP (AC532, Beyotime), phospho-PERK (AF5902; Cell Signailing, Boston, MA, USA), PERK (D11A8, Cell Signaling), IRE1 α (AI601, Beyotime), phospho-IRE1α (AF5842, Beyotime), and ATF6 (AF6243, Beyotime) at 4 °C overnight.

Techniques: Cell Culture, Control, Expressing, Gene Expression, Quantitative Proteomics, Western Blot

Journal: Genes

Article Title: STIM1–Orai1 Interaction Exacerbates LPS-Induced Inflammation and Endoplasmic Reticulum Stress in Bovine Hepatocytes through Store-Operated Calcium Entry

doi: 10.3390/genes13050874

Figure Lengend Snippet: Protein expression and location of STIM1/Orai1 in BMEC. DAPI blue fluorescence was used to label the nuclear location, FITC green fluorescence was used to label STIM1 protein, and Cy3 red fluorescence was used to label Orai1 protein. ImageJ was used to draw the line graph to show the fluorescence co-location trends.

Article Snippet: The membrane was blocked with 7% skim milk or BSA (for phosphorylated proteins) at 25 °C for 2 h. The samples were subsequently incubated at 4 °C overnight with protein-specific antibodies (all antibodies were diluted with TBST 1:1000): NF-κB p65 (AF0246, Beyotime), phosphorus-NF-κB P65 (AF5881, Beyotime), IκB α (AF2176, Beyotime), phosphoric acid IκBα (AF1870, Beyotime), TNF α (A11534, Abclonal, Wuhan, China), STIM1 (AF2614, Beyotime), Orai1 (66223-1-Ig, ProteinTech, Thermo Fisher Scientific, Waltham, MA, USA), GRP78 (AF0171, Beyotime), CHOP (AC532, Beyotime), phospho-PERK (AF5902; Cell Signailing, Boston, MA, USA), PERK (D11A8, Cell Signaling), IRE1 α (AI601, Beyotime), phospho-IRE1α (AF5842, Beyotime), and ATF6 (AF6243, Beyotime) at 4 °C overnight.

Techniques: Expressing, Fluorescence

Journal: Genes

Article Title: STIM1–Orai1 Interaction Exacerbates LPS-Induced Inflammation and Endoplasmic Reticulum Stress in Bovine Hepatocytes through Store-Operated Calcium Entry

doi: 10.3390/genes13050874

Figure Lengend Snippet: Schematic representation shows that Toll-like receptors are stimulated by LPS, activate the STIM1/Orai1-mediated SOCE channel, cause endoplasmic reticulum stress (ERS), and turn on the NF-κB signaling pathway to induce inflammation. The upregulation of STIM1 intensifies LPS-induced inflammation and endoplasmic reticulum stress (ERS), whereas the inhibition of Orai1 alleviates the inflammatory effects of LPS and ERS. LPS, lipopolysaccharide; STIM1, stromal interaction molecule 1; Orai1, calcium release-activated calcium channel protein 1; NF-κB, nuclear factor-kappa B; IκB, inhibitor of NF-κB; PERK, pancreatic elf-2 kinase pancreatic ER kinase; ATF6, activating transcription factor 6; IRE1, inositol requiring enzyme 1; GRP78, glucose-regulated protein 78; CHOP, C/EBP homologous protein; IL-6, interleukin-6; IL-8, interleukin-8; TNF-α, tumor necrosis factor-α.

Article Snippet: The membrane was blocked with 7% skim milk or BSA (for phosphorylated proteins) at 25 °C for 2 h. The samples were subsequently incubated at 4 °C overnight with protein-specific antibodies (all antibodies were diluted with TBST 1:1000): NF-κB p65 (AF0246, Beyotime), phosphorus-NF-κB P65 (AF5881, Beyotime), IκB α (AF2176, Beyotime), phosphoric acid IκBα (AF1870, Beyotime), TNF α (A11534, Abclonal, Wuhan, China), STIM1 (AF2614, Beyotime), Orai1 (66223-1-Ig, ProteinTech, Thermo Fisher Scientific, Waltham, MA, USA), GRP78 (AF0171, Beyotime), CHOP (AC532, Beyotime), phospho-PERK (AF5902; Cell Signailing, Boston, MA, USA), PERK (D11A8, Cell Signaling), IRE1 α (AI601, Beyotime), phospho-IRE1α (AF5842, Beyotime), and ATF6 (AF6243, Beyotime) at 4 °C overnight.

Techniques: Inhibition