Journal: iScience

Article Title: Small molecule SJ572946 activates BAK to initiate apoptosis

doi: 10.1016/j.isci.2022.105064

Figure Lengend Snippet: Auto-activation-impaired R127A BAK is extensively unfolded and degraded at the mitochondria (A) Titration of 15 N-labeled R127A(s) BAK with SJ572946, monitored by 15 N- 1 H HSQC 2D-NMR. (B) Per-residue CSPs induced by SJ572946 in the amide backbone of R127A(s) BAK, calculated from the spectra shown in panel (A). Dotted red line represents CSPs average +SD. (C) Mapping of the CSPs in panel (B) onto the model of R127A(s) built using the crystal structure of apo BAK identifies SJ572946 binding to the activation groove. (D) Results of liposome permeabilization assays, quantified as the AUCs of the kinetic traces from Figure S6 C, revealing the cooperation of BID BH3 peptides with SJ572946 in direct activation of R127A(s) BAK. Data are presented as the mean +SE of n = 2 experiments, each comprising n = 3 technical replicates. ∗∗∗∗p <0.0001, ∗∗∗p <0.0002, ∗∗p <0.0021, ∗p <0.0332; Tukey–Kramer one-way ANOVA. (E) Results of mitochondrial poration assays measuring cyt c release after incubation with WT BID BH3 ± SJ572946 (bottom), followed by limited proteolysis with calpain (middle) and BMH crosslinking (top) of mitochondria purified from BCL2allKO HCT116 cells constitutively expressing R127A(s) BAK. The aberrant patterns of proteolysis and crosslinking indicate that R127A(s) BAK is inactivated by adopting a conformation different from that of active BAK. Remarkably, R127A(s) BAK was susceptible to calpain proteolysis even in the absence of BH3 peptide activators. R127A(s) BAK was extensively degraded in these cells, as shown in the BMH crosslinking blots. The BMH crosslinking pattern did not change in the presence of BH3 peptide activators, consistent with BAK being in an altered unfolded conformation at the mitochondria.

Article Snippet: Briefly, cells were suspended in mannitol experimental buffer (MEB; 10 mM HEPES (pH 7.5), 150 mM mannitol, 50 mM KCl, 0.02 mM EGTA, 0.02 mM EDTA, 0.1% BSA, and 5 mM succinate) with 0.001% digitonin and treated with compound H6 alone or in combination with BIM or BID peptides (sequences Ac-MRPEIWIAQELRRIGDEFNA-NH2 and Ac-EDIIRNIARHLAQVGDSMDRY-NH2 respectively; New England Peptide).

Techniques: Activation Assay, Titration, Labeling, Residue, Binding Assay, Incubation, Purification, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Siah2–GRP78 interaction regulates ROS and provides a proliferative advantage to Helicobacter pylori -infected gastric epithelial cancer cells

doi: 10.1007/s00018-022-04437-5

Figure Lengend Snippet: Siah2 phospho-null mutant S6A disrupts mitochondrial morphology and GRP78 abundance in H. pylori-infected GECs. A Confocal micrographs showing alteration of tubulo-reticular mitochondrial morphology in uninfected and infected pcDNA3.1+, siah2 WT and siah2 S6A-transfected pDsRed2-Mito AGS stable cells. Graphs represent mitochondrial length, circularity and roundness calculated using Fiji software from the above mentioned cells. Statistical significance is determined by two-way ANOVA followed by Tukey’s post hoc analysis. Graphs represent mean ± sem. n = 3. ****P < 0.0001. B Western blot of the whole cell lysates from uninfected or infected pcDNA3.1+, siah2 WT and siah2 S6A MKN45 stably expressed cells depicting the status of GRP78, P-S6-Siah2 and Siah2. GAPDH = loading control. Bar graphs represent densitometric analysis of GRP78 status in these cells. Statistical significance is determined by using two-way ANOVA followed by Tukey’s post hoc analysis. Graphs represent mean ± sem. n = 3. **P < 0.01. C Immunoblot of the Siah2 immuno-complexes obtained from untreated and MG132-treated H. pylori-infected pcDNA3.1+, siah2 WT and siah2 S6A MKN45 stable cells showing Siah2-GRP78 interaction. Non-specific band = loading control

Article Snippet: Expression plasmids and site-directed mutagenesis Eukaryotic expression vector pcDNA3.1+ (Thermo Fisher Scientific, IL, USA), the WT human siah2 ( g ene ID: 6478) construct (by Origene Technologies, MD, USA) and pDsRed2-Mito (Clontech, CA, USA; Cat. No. 632421) were purchased. siah2 phospho-null mutants S6A and T279A were generated by site-directed mutagenesis from the WT siah2 construct using specific primers as described earlier [ 12 ]. pcDNA3.1(+)-GRP78/BiP ( grp78 ) was a gift from Dr. Richard C. Austin (Addgene plasmid #32701) [ 23 ].

Techniques: Mutagenesis, Infection, Transfection, Software, Western Blot, Stable Transfection, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Siah2–GRP78 interaction regulates ROS and provides a proliferative advantage to Helicobacter pylori -infected gastric epithelial cancer cells

doi: 10.1007/s00018-022-04437-5

Figure Lengend Snippet: Siah2 modulates ROS and interacts with GRP78 in H. pylori-infected GECs. A Micrographs depicting ROS generation in uninfected or H. pylori-infected MKN45 pcDNA3.1+, siah2 WT, siah2 phospho-null mutant S6A and T279A stably-expressed cells. Scale bars = 10 μm. The graph represents fold changes in ROS and depicts mean ± sem values. n = 3. Statistical significance is determined by two-way ANOVA followed by Tukey’s post hoc analysis. **P < 0.01, ***P < 0.001. B Above, representative western blots showing the level of P-Ser and P-Thr in uninfected or H. pylori-infected MKN45 pcDNA3.1+, siah2 WT, siah2 phospho-null mutant S6A and T279A stably-expressed cells. Below, Bar graphs represent level of P-Ser, P-Thr and Siah2 in these cells. Graphs = mean ± sem. Statistical significance is determined by two-way ANOVA followed by Tukey’s post hoc analysis (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Western blot showing the status of P-Ser, P-Thr and total Siah2 in uninfected and H. pylori-infected MKN45 pcDNA3.1+, siah2 WT, siah2 S6A and siah2 T279A stable cells. GAPDH is used as a loading control. C Western blot of the immuno-complexes representing the interaction of Siah2-GRP78 in uninfected and infected MKN45 cell whole cell lysates co-immuno-precipitated using Siah2 antibody. Non-specific band is used as the loading control. The input lanes are shown alongside to ascertain different protein expression level. D Western blot of the whole cell lysates from uninfected and infected (200 MOI) MKN45 cells showing GRP78 protein level at the indicated time points. GAPDH is used as a loading control (left). Bar graphs represent the change in GRP78 protein levels after various time intervals of infection (right). Graphs = mean ± sem. Statistical significance is determined by two-way ANOVA followed by Tukey’s post hoc analysis (n = 3). *P < 0.05. E Immunoblot representing GRP78 protein in whole cell lysates of MKN45 infected with 100, 200 and 300 MOIs for 12 h. GAPDH = loading control (left). Bar graphs represent GRP78 protein status at various MOIs (right). Graphs = mean ± sem. Statistical significance is determined by one-way ANOVA (n = 3). *P < 0.05; ***P < 0.001. F Western blots depicting GRP78 status in MKN45 cells infected with H. pylori cag PAI ( +) 26,695 and cag PAI (-) 8–1 strains (left). Bar graphs showing the cag PAI-independent decrease of GRP78 (right). Graphs = mean ± sem. Statistical significance is determined by one-way ANOVA (n = 3). ***P < 0.001

Article Snippet: Expression plasmids and site-directed mutagenesis Eukaryotic expression vector pcDNA3.1+ (Thermo Fisher Scientific, IL, USA), the WT human siah2 ( g ene ID: 6478) construct (by Origene Technologies, MD, USA) and pDsRed2-Mito (Clontech, CA, USA; Cat. No. 632421) were purchased. siah2 phospho-null mutants S6A and T279A were generated by site-directed mutagenesis from the WT siah2 construct using specific primers as described earlier [ 12 ]. pcDNA3.1(+)-GRP78/BiP ( grp78 ) was a gift from Dr. Richard C. Austin (Addgene plasmid #32701) [ 23 ].

Techniques: Infection, Mutagenesis, Stable Transfection, Western Blot, Control, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Siah2–GRP78 interaction regulates ROS and provides a proliferative advantage to Helicobacter pylori -infected gastric epithelial cancer cells

doi: 10.1007/s00018-022-04437-5

Figure Lengend Snippet: Proteasomal degradation of GRP78 is Siah2-mediated in H. pylori-infected GECs. A Confocal micrographs illustrating the levels of GRP78 protein in uninfected and infected pcDNA3.1+ and siah2 WT MKN45 stable cells. Scale bars represent 5 μm (left). Bar graphs represent fold change in the immunofluorescence of GRP78 (right). Graphs = mean ± sem. n = 3. Statistical significance is determined by two-way ANOVA followed by Tukey’s post hoc analysis. ****P < 0.0001. B Micrographs representing GRP78 protein levels in untreated, DMSO-treated and MG132-treated uninfected or infected pcDNA3.1+ and siah2 WT stably expressing MKN45 cells. Scale bars represent 5 μm. Graphs represent fold change in GRP78 levels. Graphs = mean ± sem. n = 3. Statistical significance is determined by three-way ANOVA followed by Tukey’s post hoc analysis. ****P < 0.0001. C Western blot representing ubiquitin aggregates in H. pylori-infected and MG132-treated MKN45 cells. Arrow indicates the position of ubiquitinated-GRP78 (left). Re-probed blots showing GRP78 status in H. pylori-infected and MG132-treated MKN45 cells (right). GAPDH = loading control. Bar graph represents GRP78 protein rescue in MG132-treated and H. pylori-infected MKN45 cells. Statistical significance is determined by one-way ANOVA (right). Graphs = mean ± sem. n = 3. **P < 0.01. D Western blot depicting autophagy-independent GRP78 decrease and Siah2 increase as assessed by infecting MKN45 cells in the presence or the absence of 10 nM autophagy inhibitor bafilomycin A1. GAPDH is used as the loading control. E Western blot of the whole cell lysates from uninfected and infected control or siah2 siRNA-transfected MKN45 cells depicting the levels of GRP78 and Siah2. GAPDH = loading control. Bar graphs represent GRP78 protein in the similar setup. Statistical significance is determined by two-way ANOVA followed by Tukey’s post hoc analysis. Graphs = mean ± sem. n = 3. **P < 0.01, ***P < 0.001

Article Snippet: Expression plasmids and site-directed mutagenesis Eukaryotic expression vector pcDNA3.1+ (Thermo Fisher Scientific, IL, USA), the WT human siah2 ( g ene ID: 6478) construct (by Origene Technologies, MD, USA) and pDsRed2-Mito (Clontech, CA, USA; Cat. No. 632421) were purchased. siah2 phospho-null mutants S6A and T279A were generated by site-directed mutagenesis from the WT siah2 construct using specific primers as described earlier [ 12 ]. pcDNA3.1(+)-GRP78/BiP ( grp78 ) was a gift from Dr. Richard C. Austin (Addgene plasmid #32701) [ 23 ].

Techniques: Infection, Immunofluorescence, Stable Transfection, Expressing, Western Blot, Ubiquitin Proteomics, Control, Transfection

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Siah2–GRP78 interaction regulates ROS and provides a proliferative advantage to Helicobacter pylori -infected gastric epithelial cancer cells

doi: 10.1007/s00018-022-04437-5

Figure Lengend Snippet: Decreased GRP78 and increased Siah2 are features of Helicobacter-infected gastric epithelia. A Human antral metastatic GC biopsy tissues depicting the status of GRP78, P-Ser6-Siah2 and Siah2. B Uninfected and H. felis-infected mouse gastric tissue sections representing GRP78 decrease and P-Ser6-Siah2 or Siah2 increase. Scale bars = 100 μm

Article Snippet: Expression plasmids and site-directed mutagenesis Eukaryotic expression vector pcDNA3.1+ (Thermo Fisher Scientific, IL, USA), the WT human siah2 ( g ene ID: 6478) construct (by Origene Technologies, MD, USA) and pDsRed2-Mito (Clontech, CA, USA; Cat. No. 632421) were purchased. siah2 phospho-null mutants S6A and T279A were generated by site-directed mutagenesis from the WT siah2 construct using specific primers as described earlier [ 12 ]. pcDNA3.1(+)-GRP78/BiP ( grp78 ) was a gift from Dr. Richard C. Austin (Addgene plasmid #32701) [ 23 ].

Techniques: Infection

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Siah2–GRP78 interaction regulates ROS and provides a proliferative advantage to Helicobacter pylori -infected gastric epithelial cancer cells

doi: 10.1007/s00018-022-04437-5

Figure Lengend Snippet: GRP78 is crucial in the regulation of ROS in H. pylori-infected GECs. A Micrographs depicting ROS generation in uninfected or H. pylori-infected (200 MOI, 12 h) empty vector and grp78 stably-expressing AGS cells. Cat+/Cat− refer to catalase-treated/catalase-untreated cells, respectively. Western blots represent the status of GRP78 in uninfected or infected empty vector and grp78 stably-expressing AGS cells. GAPDH = loading control. Graphs represent fold change in ROS generation. B Fluorescence microscopy images representing ROS generation in AGS cells transfected with control and grp78 siRNA for 36 h followed by 12 h of H. pylori infection. Cat+/Cat− refer to catalase-treated/catalase-untreated cells, respectively. Western blots represent the status of GRP78 protein expression after grp78 suppression and H. pylori infection. GAPDH = loading control. Fold change in ROS generation is represented by bar graphs. C Micrographs depicting ROS generation in uninfected or infected control and siah2 suppressed AGS cells. Cat+/Cat− refer to catalase-treated/catalase-untreated cells, respectively. Western blots represent the status of Siah2 protein expression in the same experimental setup. GAPDH = loading control. Graphs represent fold change in ROS generation. For all images, scale bars = 10 μm. Graphs = mean ± sem. n = 3. Statistical significance is determined by three-way ANOVA followed by Tukey’s post hoc analysis. **P < 0.01, ***P < 0.001, ****P < 0.0001

Article Snippet: Expression plasmids and site-directed mutagenesis Eukaryotic expression vector pcDNA3.1+ (Thermo Fisher Scientific, IL, USA), the WT human siah2 ( g ene ID: 6478) construct (by Origene Technologies, MD, USA) and pDsRed2-Mito (Clontech, CA, USA; Cat. No. 632421) were purchased. siah2 phospho-null mutants S6A and T279A were generated by site-directed mutagenesis from the WT siah2 construct using specific primers as described earlier [ 12 ]. pcDNA3.1(+)-GRP78/BiP ( grp78 ) was a gift from Dr. Richard C. Austin (Addgene plasmid #32701) [ 23 ].

Techniques: Infection, Plasmid Preparation, Stable Transfection, Expressing, Western Blot, Control, Fluorescence, Microscopy, Transfection

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Siah2–GRP78 interaction regulates ROS and provides a proliferative advantage to Helicobacter pylori -infected gastric epithelial cancer cells

doi: 10.1007/s00018-022-04437-5

Figure Lengend Snippet: Mitochondrial localization of GRP78 and Siah2. A Confocal microscopy images from uninfected and infected (200 MOI, 12 h) pDsRed2-Mito-expressing AGS stable cells depicting the mitochondrial expression of GRP78, P-S6-Siah2 and Siah2. Scale bars represent 50 μm. B Western blots from the cytoplasmic and the mitochondrial fractions of uninfected and infected MKN45 cells depicting the expression of GRP78, P-S6-Siah2 and Siah2. CoxIV is used as a loading control for the mitochondrial fraction and GAPDH for the cytoplasmic fraction, respectively. Bar graphs represent changes in the expression of GRP78, P-S6-Siah2 and Siah2 in the cytoplasmic and mitochondrial cellular fractions. Graphs = mean ± sem. n = 3. Statistical significance is determined by two-tailed unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001. C Western blots form the whole cell lysates of uninfected and infected pcDNA3.1+, siah2 WT, siah2 S6A and siah2 T279A MKN45 stable cells representing specificity of the P-S6-Siah2 antibody indicated by the lowest level in S6A cells

Article Snippet: Expression plasmids and site-directed mutagenesis Eukaryotic expression vector pcDNA3.1+ (Thermo Fisher Scientific, IL, USA), the WT human siah2 ( g ene ID: 6478) construct (by Origene Technologies, MD, USA) and pDsRed2-Mito (Clontech, CA, USA; Cat. No. 632421) were purchased. siah2 phospho-null mutants S6A and T279A were generated by site-directed mutagenesis from the WT siah2 construct using specific primers as described earlier [ 12 ]. pcDNA3.1(+)-GRP78/BiP ( grp78 ) was a gift from Dr. Richard C. Austin (Addgene plasmid #32701) [ 23 ].

Techniques: Confocal Microscopy, Infection, Expressing, Western Blot, Control, Two Tailed Test

Journal: Diabetes

Article Title: Functional Gly297Ser Variant of the Physiological Dysglycemic Peptide Pancreastatin Is a Novel Risk Factor for Cardiometabolic Disorders.

doi: 10.2337/db21-0289

Figure Lengend Snippet: Figure 6—In vitro interaction studies between human PST peptides and GRP78. A: Spectrophotometric Malachite Green-phosphate assay was performed to compare the effect of 2 mmol/L PST-WT and PST-297S on inhibition of GRP78 ATPase activity. The different groups were compared using one-way ANOVA followed by Tukey multiple-comparisons test; n 5 4, one-way ANOVA, F 5 30.93, P < 0.0001. B: Human HepG2 hepatocytes were treated with 100 nmol/L PST-WT or PST-297S along with 5 mg/mL tunicamycin or 5 mg/mL tunicamycin alone for 24 h. Changes in GRP78 expression following this treatment were visualized using an immunoblot with anti-GRP78 antibody (control: anti–b-actin). C: Quantitative analysis of the immunoblot in B was performed using one-way ANOVA followed by Tukey multiple-comparisons test; n 5 5, one-way ANOVA, F 5 35.29, P < 0.0001. D: Western blot for GRP78 expression posttransfection of HEK-293 cells with increasing amounts of GRP78-overexpressing construct: lane 1, 0 mg; lane 2, 0.5 mg; lane 3, 1 mg; lane 4, 2 mg; and lane 5, 3 mg of GRP78-overexpressing construct. E: Competitive binding assay was performed to assess the ability of PST-WT or PST- 297S peptides to displace labeled [125]I-Tyr PST by adding increasing concentrations (0, 10 pmol/L through 1 mmol/L) of the PST peptides along with 100 nCi of labeled PST to 10 mg of isolated plasma membrane. Displacement was calculated as a percentage of receptors in the control samples that did not contain any competing peptides. F: Quantitative analysis of the graph in E was performed by comparing the AUC of the displacement curves of PST-WT and PST-297S using an unpaired two-tailed Student t test (n 5 4).

Article Snippet: GRP78- and IR-overexpression constructs were generated by cloning GRP78 cDNA (obtained from catalog no. 32701; Addgene, Watertown, MA) and IR cDNA (a kind gift from Dr. Frederick M. Stanley [24]) into pcDNA 3.1.

Techniques: In Vitro, Inhibition, Activity Assay, Expressing, Western Blot, Control, Construct, Competitive Binding Assay, Labeling, Isolation, Clinical Proteomics, Membrane, Two Tailed Test

Journal: Diabetes

Article Title: Functional Gly297Ser Variant of the Physiological Dysglycemic Peptide Pancreastatin Is a Novel Risk Factor for Cardiometabolic Disorders.

doi: 10.2337/db21-0289

Figure Lengend Snippet: Figure 7—A schematic representation of the plausible mechanistic basis for increased cardiometabolic disease risk associated with the PST 297Ser allele. Occurrence of a nonsynonymous genetic variation within the PST region results in the PST p.Gly297Ser variant. PST- 297S peptide differs from PST-WT in its secondary structure (especially a-helical content). Structural differences between PST-WT and PST-297S peptides and their consequent differential interactions with GRP78 and IR may cause enhanced potencies of the PST-297S peptide for inhibition of insulin-stimulated glucose uptake and activation of the gluconeogenesis pathway. These and associated cellular/ molecular processes may elevate the levels of plasma glucose, HbA1c, and catecholamines in the carriers of the PST 297Ser allele, thereby increasing their risk for T2D, HTN, CAD, and MS.

Article Snippet: GRP78- and IR-overexpression constructs were generated by cloning GRP78 cDNA (obtained from catalog no. 32701; Addgene, Watertown, MA) and IR cDNA (a kind gift from Dr. Frederick M. Stanley [24]) into pcDNA 3.1.

Techniques: Variant Assay, Inhibition, Activation Assay, Clinical Proteomics

Journal: Scientific Reports

Article Title: Extracellular calcium alters calcium-sensing receptor network integrating intracellular calcium-signaling and related key pathway

doi: 10.1038/s41598-021-00067-2

Figure Lengend Snippet: The schematic model of CaSR mediated signaling, biosynthesis and ER quality control of CaSR invoked through Ca 2+ ext perturbation. Identified proteins from the experiment are colored respectively with fold-enrichment (log 2 (Ca 2+ /EGTA)-positive controls). ( A ) (I) CaSR is activated with 4 mM Ca 2+ ex . (II) Ca 2+ ex /CaSR mediated G-protein signaling through phospho-lipase C(PLC) activation and subsequently, IP 3 invoked release of Ca 2+ from the ER. Ca 2+ dependent Gα i invoked inhibition of acetyl cyclase is induced. ( B ) Alteration in Ca 2+ ER induce amplification of CaSR interactomes. Biosynthesis and processing in the ER. (III) Co-translational translocation: The newly synthesized polypeptide bound to the SRP is directed to the ER membrane to the Sec complex by the SRP receptor, then processed by oligosaccharyl transferases (OST: DDOST and RPN1/2). GRP78 chaperones CaSR polypeptide to ER lumen where glucosidases GANAB allows for the removal of the outermost glucose residues. (IV) Calnexin cycle CANX and/or CRT along with GRP78 promotes folding and PDIA3 catalyzes disulfide formation. (V) ER-exit Properly folded CaSR traffics from ER to the Golgi assisted by VAPA and VAPB as well as p24A. Rab6 assists in retrograde trafficking from Golgi to the ER. 14-3-3 allows the CaSR to remain in the ER in the absence of Ca 2+ . UGGT acts as a check- point for improperly folded CaSR. [VI] Degradation pathway: Terminally misfolded CaSR undergoes ERAD and is ubiquitinated through STUB1. (VII) Endosomal degradation: CaSR from the plasma membrane can be endocytosed assisted by Rab5, Rab18 and CLTC. ( C ) Ca 2+ ER release via CaSR monitored using G-CatchER + (green line). Activated CaSR mediated Ca 2+ i mobilized from ER measured using Fura-red (red line).

Article Snippet: Empty pcDNA3.1, human CaSR with FLAG-tag (FLAG-hCaSR) (between Asp 371 and Thr 372 ) in pcDNA3.1 and EGFP-CaSR (provided by Dr. Chen Zhang, La Jolla Institute of Allergy and Immunology, CA) were used for transfection for negative and positive controls, respectively. pEGFPC1-hVAPA and pcDNA3.1 GRP78 were obtained from Addgene. mCherry-VAPA was constructed from pEGFPC1-hVAPA (Addgen) and mCherry in pcDNA3.1.

Techniques: Control, Activation Assay, Inhibition, Amplification, Translocation Assay, Synthesized, Membrane, Clinical Proteomics

Journal: Scientific Reports

Article Title: Extracellular calcium alters calcium-sensing receptor network integrating intracellular calcium-signaling and related key pathway

doi: 10.1038/s41598-021-00067-2

Figure Lengend Snippet: Interactions of CaSR with assistant proteins, VAPA and GRP78, and Ca 2+ ext modulated CaSR-VAPA interdependent cell membrane expression. Co-immunoprecipitation experiments with either Ca 2+ or EGTA treatments using anti-FLAG antibody on total cell extracts from HEK293 transfected with FLAG-CaSR and VAPA or GRP78 pcDNA3.1 followed by western blot on 50 µg total extract. Analysis with anti-CaSR antibody (( A,B ) insert; second and fifth panel), anti-VAPA antibody (( A ) insert, first and fourth panel), anti-GRP78 (( B ) insert, first and fourth panel) or anti-GAPDH (as control, ( A,B ), third panel) was carried out in triplicate (one representative blot is shown) and is represented in bar graph for output VAPA ( A ) and output GRP78 ( B ). Representative confocal imaging ( C ) showing colocalization of VAPA (red) (in physiological condition (first panel) and 4 mM Ca 2+ (second panel)), or GRP78 (red) (in physiological condition (third panel) and 4 mM Ca 2+ (fourth panel)) with CaSR (green) and ER marker (calreticulin, magenta) along the colocalized region (yellow). Scale bar, 2.5 µm. Comparison of pixel intensity correlation of CaSR and ER with VAPA ( D ) or GRP78 ( E ) in confocal images conducted at the regions between cell nuclei and cell membrane boundaries in single cell image sections of Cos7 cells obtained after treatments with physiological Ca 2+ (DMEM with 2.2 mM) vs 4 mM Ca 2+ . Statistical calculations (Mean ± SD) were determined from five independent cells from different slides obtained with separate transfections (scatter dot). Representative dual-color TIRF images of cells expressing both VAPA and CaSR (( F ), upper panel), expressing only VAPA (( F ), middle panel), and expressing only CaSR (( F ), lower panel). Scale bar, 10 µm. Relative fluorescence intensity changes of membrane expressed CaSR signal in cells with (black line) and without (red line) co-expressing VAPA, represented by a single cell ( G ) and an average representation with n > 5 ( H ). The density of VAPA spots near the cell membrane in cells with (black line) and without (red line) co-expressing CaSR, represented by a single cell ( I ) and an average representation with n > 7 ( J ).

Article Snippet: Empty pcDNA3.1, human CaSR with FLAG-tag (FLAG-hCaSR) (between Asp 371 and Thr 372 ) in pcDNA3.1 and EGFP-CaSR (provided by Dr. Chen Zhang, La Jolla Institute of Allergy and Immunology, CA) were used for transfection for negative and positive controls, respectively. pEGFPC1-hVAPA and pcDNA3.1 GRP78 were obtained from Addgene. mCherry-VAPA was constructed from pEGFPC1-hVAPA (Addgen) and mCherry in pcDNA3.1.

Techniques: Membrane, Expressing, Immunoprecipitation, Transfection, Western Blot, Control, Imaging, Marker, Comparison, Fluorescence