Journal: Frontiers in Immunology

Article Title: Combined p14ARF and Interferon-β Gene Transfer to the Human Melanoma Cell Line SK-MEL-147 Promotes Oncolysis and Immune Activation

doi: 10.3389/fimmu.2020.576658

Figure Lengend Snippet: Emission of immunogenic cell death markers induced by combined adenoviral p14ARF + IFNβ gene transfer. SK-MEL-147 cells transduced as previously described, incubated for 48h h before cells and supernatants were collected for ICD assays. (A) Calreticulin exposure was assessed by flow cytometry after specific antibody staining. Representative dot plots and a bar graph showing the mean and standard deviation from three independent tests with three technical replicates each. (B) Supernatant from the same cultures were collected and evaluated for ATP secretion using a luciferase-based assay (RLU, relative light units). Data represent the mean and standard deviation from at least three independent experiments. (C) Detection of secreted IFNβ protein from cell supernatant by ELISA. Data represent the mean and standard deviation from at least three independent experiments. For both (A–C) , statistical analyses were performed using one-way ANOVA test followed by the Bonferroni post-test. *p < 0.05, **p < 0.005, and ***p < 0.0005.

Article Snippet: For the evaluation of calreticulin exposure through flow cytometry, cells were probed with rabbit anti-calreticulin antibody (Novus Biologicals, Littleton, CO, USA), followed by the Alexa488-conjugated anti-rabbit secondary antibody (Thermo Fisher Scientific).

Techniques: Incubation, Flow Cytometry, Staining, Standard Deviation, Luciferase, Enzyme-linked Immunosorbent Assay

Journal: Circulation research

Article Title: Destabilization of AT(1) receptor mRNA by calreticulin.

doi: 10.1161/hh0102.102503

Figure Lengend Snippet: Figure 3. Identification of the AT1 recep- tor mRNA binding protein calreticulin. A, Polysomal proteins were isolated and subjected to sequential anionic exchange chromatographies. Eluted fractions were monitored with crosslink assays (ribo- probe AT1 receptor mRNA 1864 to 2213). Proteins on lanes 8 and 9 (right gel) were separated by electrophoresis, the corre- sponding protein was excised, eluted, and characterized by MALDI. N indicates no protein; P, probe without digestion. B, Amino acid sequences of protein frag- ments derived from fingerprinting analy- sis following MALDI. Alignment with known protein sequences using Pro- Found searches revealed homology with calreticulin. C, Phosphorylated calreticu- lin binds to the AT1 receptor mRNA. Recombinant calreticulin (cal) was phos- phorylated with Src kinase, JNK kinase, -insulin receptor kinase (-IR kinase), or autophosphorylated. Binding of calreticu- lin to the AT1 receptor riboprobe bases 1864 to 2213 was monitored by UV crosslink assay. When UV crosslinking of calreticulin to the AT1 receptor mRNA is omitted, no binding activity is visualized (negative control). D, Calreticulin was phosphorylated with Src kinase, JNK kinase, -IR kinase, or autophosphorylat- ed in the presence of 32P-ATP. Reactions were separated on a acrylamide gel. Phosphorylated calreticulin was detected by autoradiography. E, Phosphorylated calreticulin was incubated and crosslinked with the AT1 receptor ribo- probe bases 1864 to 2213 in the pres- ence of the AT1 receptor mRNA competi- tors bases 1864 to 2213, 2175 to 2195, and the unspecific mRNA competitor GAPDH. The binding pattern reveals that calreticulin binds the AT1 receptor mRNA at bases 2175 to 2195.

Article Snippet: After centrifugation, supernatants were incubated with an anti-calreticulin antibody (Novus Biologicals).

Techniques: Binding Assay, Isolation, Electrophoresis, Derivative Assay, Recombinant, Activity Assay, Negative Control, Acrylamide Gel Assay, Autoradiography, Incubation

Journal: Circulation research

Article Title: Destabilization of AT(1) receptor mRNA by calreticulin.

doi: 10.1161/hh0102.102503

Figure Lengend Snippet: Figure 4. VSMCs were stimulated with 100 nmol/L angiotensin II for 0, 2, 5, 10, 15, and 20 minutes. Cell protein was homoge- nized, calreticulin immunoprecipitated, and the phosphorylation of calreticulin was tested in a Western blot with an anti- phosphotyrosine antibody. Angiotensin II led to a profound phosphorylation of calreticulin.

Article Snippet: After centrifugation, supernatants were incubated with an anti-calreticulin antibody (Novus Biologicals).

Techniques: Immunoprecipitation, Phospho-proteomics, Western Blot

Journal: Circulation research

Article Title: Destabilization of AT(1) receptor mRNA by calreticulin.

doi: 10.1161/hh0102.102503

Figure Lengend Snippet: Figure 5. Calreticulin destabilizes the AT1 receptor mRNA. A, Full- length calreticulin cDNA cloned in the expression vector pcDNA3 (cal) or the insert-less pcDNA3 (control) were transfected in VSMCs via electroporation technique. Overexpression and transfection efficiency above 20% was assured. After 48 hours, AT1 receptor mRNA expres- sion was monitored with Northern blotting. Calreticulin overexpression led to a significant downregulation of AT1 receptor mRNA. 18S RNA remained unaltered. Densitometric analysis of 5 separate experiments. B, Either recombinant phosphorylated calreticulin (AT1-Cal) or the kinase reaction without calreticulin (AT1-control) were added to an in vitro decay assay including the in vitro transcribed AT1 receptor mRNA. Degradation of AT1 receptor mRNA was assessed by real- time PCR. As internal control, a GAPDH mRNA was included in the reaction and concomitantly quantified (GAPDH-Cal) (n3, meanSEM; *P0.05). C, Cells were transfected with either an insert- less pcDNA3 vector (control and angII) or an antisense calreticulin construct (AS-Cal). The effect of antisense calreticulin and sense calre- ticulin (S-Cal). Construct transfection was monitored by Western blots (insert). Twenty-four hours later VSMCs were incubated with vehicle (control) or 1 mol/L angiotensin II (angII) for 4 hours. AT1 receptor and GAPDH mRNA were quantified by real-time PCR (n5, meanSEM; *P0.05).

Article Snippet: After centrifugation, supernatants were incubated with an anti-calreticulin antibody (Novus Biologicals).

Techniques: Clone Assay, Expressing, Plasmid Preparation, Control, Transfection, Electroporation, Over Expression, Northern Blot, Recombinant, In Vitro, Real-time Polymerase Chain Reaction, Construct, Western Blot, Incubation

Journal: eLife

Article Title: Biallelic TANGO1 mutations cause a novel syndromal disease due to hampered cellular collagen secretion

doi: 10.7554/eLife.51319

Figure Lengend Snippet: Immunofluorescence images of U2OS cells, transiently transfected with WT TANGO1-HA or Ex8-HA. Representative images of three independent experiments. ( A ) Cells were probed with anti-HA (green), anti-Sec16A (red) and anti-Calreticulin (blue) antibodies. Scale bar 10 μm. ( B ) Merged images of TANGO1-HA or Ex8-HA (green) and sec16A (red) and a plot comparing Manders’ overlap coefficient of HA with sec16A in TANGO1- or Ex8-expressing U2OS cells. ( C ) Merged images of TANGO1-HA or Ex8-HA (green) with calreticulin (red) and a plot comparing Manders’ overlap coefficient of HA and calreticulin in TANGO1- or Ex8-expressing U2OS cells. Student’s t test was performed to compare the Manders’ overlap coefficients, p values are shown.

Article Snippet: Antibodies used in Western blotting and immunofluorescence microscopy were as follows: TANGO1 (Sigma-Aldrich); beta-tubulin (Sigma-Aldrich); calreticulin (Novus Biologicals, Centennial, Colorado, USA); Calnexin (Abcam, Cambridge, United Kingdom); antitrypsin Ab-1 (NeoMarkers, Fremont, California, USA); collagen I and collagen IV (Abcam, Cambridge, United Kingdom); collagen XII (Santa Cruz Biotechnology, Dallas, Texas, USA); Sec16A (Sigma-Aldrich); rat hemagglutinin (Roche) or mouse hemagglutinin (Santa Cruz Biotechnology, Dallas, Texas, USA).

Techniques: Immunofluorescence, Transfection, Expressing