Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Evolutionary conservation and sequence homology analysis of calreticulin across multiple species. ( A ) Phylogenetic analysis of calreticulin amino acid sequences from different species. The neighbor-joining phylogenetic tree was constructed using the bootstrap method in MEGA version 10.2.2 with 1000 bootstrap replicates. ( B ) Sequence homology analysis of calreticulin amino acid sequences among various species. The matrix shown represents a bidirectional pairwise comparison of calreticulin sequences. The upper triangular region displays the percentage of sequence identity (% identity), while the lower triangular region shows the corresponding percentage of sequence divergence (% divergence) between each pair of species. Black squares along the diagonal indicate self-alignments, where sequence identity is 100% and divergence is 0%.

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Sequencing, Construct, Comparison

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Distribution of calreticulin in different goat organs. ( A ) Immunohistochemical staining illustrating the localization of calreticulin in the nasal mucosa, pharynx, trachea, lung, heart, liver, spleen, and small intestine of goats. Bars = 50 μm. ( B ) Analysis of calreticulin protein expression levels via Western blot in the nasal mucosa, pharynx, trachea, lung, heart, liver, spleen, and small intestine of goats. ( C ) The band obtained by Western blot was analyzed by gray value, and the result was expressed as the gray value ratios of the CALR/GAPDH. All data shown are the mean ± SD from three independent experiments. Original Western blot images can be found in .

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Immunohistochemical staining, Staining, Expressing, Western Blot

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Expression and purification of recombinant goat calreticulin using the Pichia pastoris expression system. ( A ) Schematic representation of the recombinant plasmid PpIC9K-CALR. ( B ) Agarose gel electrophoresis was performed to analyze the original recombinant plasmid (lane 1), the plasmid digested with EcoRI alone (lane 2), and the plasmid digested with both EcoRI and NotI (lane 3). ( C ) SDS-PAGE analysis of recombinant calreticulin expression in methanol-induced positive transformants, stained with Coomassie Brilliant Blue: Lane 1—culture supernatant of methanol-induced transformants; Lane 2—flow-through during Ni-NTA affinity purification; Lane 3—wash fraction; Lane 4—eluted protein with 200 mM imidazole; Lane 5—desalted and concentrated protein following ultrafiltration. ( D ) Western blot analysis confirming the identity of purified recombinant goat calreticulin (lane order as in panel ( C )). ( E ) SEC-HPLC analysis of the molecular weight and purity of yeast-secreted recombinant goat calreticulin. Original Western blot images can be found in .

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Expressing, Purification, Recombinant, Plasmid Preparation, Agarose Gel Electrophoresis, SDS Page, Staining, Affinity Purification, Western Blot, Molecular Weight

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Antibacterial activity of recombinant goat calreticulin. ( A – C ) CFU assays were performed to evaluate the inhibitory effects of calreticulin against Escherichia coli , Salmonella typhimurium , and Pasteurella multocida . ( D – F ) Growth curve analysis of Escherichia coli , Salmonella typhimurium , and Pasteurella multocida in the presence of calreticulin to assess its impact on bacterial proliferation. All data are presented as mean ± SD from three independent experiments. Statistical significance was determined using one-way ANOVA. ns, no significance; *** p < 0.001.

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Activity Assay, Recombinant

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Recombinant goat calreticulin binds to and agglutinates bacteria. ( A – C ) ELISA analysis of bacterial binding: Escherichia coli , Salmonella typhimurium , and Pasteurella multocida (1 × 10 7 CFU/mL) were immobilized on microtiter plates and incubated with either CaCl 2 (10 mM), His-tag peptide (100 μg/mL), His-tag peptide (100 μg/mL) + CaCl 2 (10 mM), calreticulin (100 μg/mL), or calreticulin (100 μg/mL) + CaCl 2 (10 mM). Binding of calreticulin was detected using anti-His tag antibodies. ( D ) Western blot analysis of bacterial pellets after incubation with calreticulin (100 μg/mL) to confirm binding to Escherichia coli , Salmonella typhimurium , and Pasteurella multocida . ( E ) ELISA analysis of calreticulin binding activity to LPS. ( F ) Log-phase Escherichia coli , Salmonella typhimurium , and Pasteurella multocida were labeled with CFSE and incubated with calreticulin (100 μg/mL) ± 10 mM Ca 2+ at 37 °C for 2 h. Bacterial agglutination was visualized by fluorescence microscopy. Bars = 100 μm. All data are expressed as the mean ± SD from three independent experiments. Statistical significance was assessed using one-way ANOVA. ns, no significance; *** p < 0.001. Original Western blot images can be found in .

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Recombinant, Bacteria, Enzyme-linked Immunosorbent Assay, Binding Assay, Incubation, Western Blot, Activity Assay, Labeling, Agglutination, Fluorescence, Microscopy

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Intranasal infection with Pasteurella multocida upregulates calreticulin expression in the respiratory tissues of the lambs. Thirty-day-old lambs were intranasally challenged with Pasteurella multocida and euthanized 24 h post-infection, after which respiratory tract tissues, including the nasal cavity, trachea, and lung, were collected for further analysis. ( A ) Immunohistochemical staining showing calreticulin expression in the nasal mucosa, trachea, and lungs following infection. Bars = 20 μm. ( B ) Quantitative analysis of immunohistochemical staining based on gray value measurements. ( C ) RT-qPCR analysis of calreticulin mRNA expression in different respiratory tissues after Pasteurella multocida infection. ( D ) Western blot analysis of calreticulin protein levels in infected respiratory tissues. ( E ) Densitometric analysis of Western blot results. All data are presented as mean ± SD from three independent experiments. Statistical significance was evaluated using one-way ANOVA. ** p < 0.01; *** p < 0.001. Original Western blot images can be found in .

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Infection, Expressing, Immunohistochemical staining, Staining, Quantitative RT-PCR, Western Blot

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Intranasal administration of calreticulin alleviates Pasteurella multocida -induced pathological injury and promotes bacterial clearance in the lambs. Thirty-day-old lambs were intranasally infected with Pasteurella multocida ; 6 h post-infection, 1 mL of recombinant calreticulin (2.5 mg/mL) was administered intranasally. Lambs were euthanized and necropsied at 24 h post-infection for sample collection. ( A ) Histopathological evaluation of nasal mucosa ( a – c ), trachea ( d – f ), and lung tissues ( g – i ) using H&E staining. Bars = 20 μm. ( B ) Average injury scores calculated for all lambs ( n = 3 replicates/group). ( C ) In situ hybridization with a Pasteurella multocida -specific fluorescent probe to detect bacterial load in nasal cavity ( a , b ), tracheal ( c , d ), and lung ( e , f ) tissue sections. Bars = 100 μm. ( D ) Quantification of fluorescence intensity derived from in situ hybridization results. All data are presented as mean ± SD from three independent experiments. Statistical significance was assessed using one-way ANOVA. *** p < 0.001.

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Infection, Recombinant, Staining, In Situ Hybridization, Fluorescence, Derivative Assay

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Distribution of calreticulin in different goat organs. ( A ) Immunohistochemical staining illustrating the localization of calreticulin in the nasal mucosa, pharynx, trachea, lung, heart, liver, spleen, and small intestine of goats. Bars = 50 μm. ( B ) Analysis of calreticulin protein expression levels via Western blot in the nasal mucosa, pharynx, trachea, lung, heart, liver, spleen, and small intestine of goats. ( C ) The band obtained by Western blot was analyzed by gray value, and the result was expressed as the gray value ratios of the CALR/GAPDH. All data shown are the mean ± SD from three independent experiments. Original Western blot images can be found in .

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Immunohistochemical staining, Staining, Expressing, Western Blot

Journal: PLoS ONE

Article Title: Differential Regulation of Cell-Cell Contact, Invasion and Anoikis by hScrib and hDlg in Keratinocytes

doi: 10.1371/journal.pone.0040279

Figure Lengend Snippet: Cells were grown to 80% confluency and then extracted into cytosolic (F1), membrane (F2), nuclear (F3) and cytoskeletal (F4) fractions. These were then analysed for the distribution of β-catenin and E-cadherin by western blotting. Controls for the integrity of the fractions were vimentin for the cytoskeletal fraction, p84 for the nuclear fraction, α-tubulin for the cytosolic fraction and transferrin receptor for the membrane fraction.

Article Snippet: The following commercial antibodies were used at the dilution indicated: anti-hScrib goat polyclonal antibody (Santa Cruz, CA, USA; Western Blot 1∶1000), anti-hDlg1 mouse monoclonal antibody (Santa Cruz, CA, USA; Western Blot 1∶1000), anti-γ-tubulin mouse monoclonal antibody (Abcam; Western Blot 1∶5000), anti-ZO-1 rabbit polyclonal antibody (Santa Cruz; CA,USA; Immuofluorescence 1∶100), anti β-catenin mouse monoclonal (Santa Cruz; CA, USA; Western Blot 1∶500), anti E-Cadherin rabbit polyclonal (Santa Cruz; CA, USA; Western Blot 1∶500), anti- Vimentin mouse monoclonal (Santa Cruz; CA, USA; Western Blot 1∶1000), anti-p84 mouse monoclonal (Santa Cruz; CA, USA; Western Blot 1∶1000), anti-transferrin receptor mouse monoclonal (Santa Cruz, USA; Wesern Blot 1∶1000).

Techniques: Membrane, Western Blot

Journal: PLoS ONE

Article Title: Differential Regulation of Cell-Cell Contact, Invasion and Anoikis by hScrib and hDlg in Keratinocytes

doi: 10.1371/journal.pone.0040279

Figure Lengend Snippet: Cells were grown to 80% confluency and then extracted into cytosolic (F1), membrane (F2), nuclear (F3) and cytoskeletal (F4) fractions. These were then analysed for the distribution of β-catenin and E-cadherin by western blotting. Controls for the integrity of the fractions were vimentin for the cytoskeletal fraction, p84 for the nuclear fraction, α-tubulin for the cytosolic fraction and transferrin receptor for the membrane fraction.

Article Snippet: The following commercial antibodies were used at the dilution indicated: anti-hScrib goat polyclonal antibody (Santa Cruz, CA, USA; Western Blot 1∶1000), anti-hDlg1 mouse monoclonal antibody (Santa Cruz, CA, USA; Western Blot 1∶1000), anti-γ-tubulin mouse monoclonal antibody (Abcam; Western Blot 1∶5000), anti-ZO-1 rabbit polyclonal antibody (Santa Cruz; CA,USA; Immuofluorescence 1∶100), anti β-catenin mouse monoclonal (Santa Cruz; CA, USA; Western Blot 1∶500), anti E-Cadherin rabbit polyclonal (Santa Cruz; CA, USA; Western Blot 1∶500), anti- Vimentin mouse monoclonal (Santa Cruz; CA, USA; Western Blot 1∶1000), anti-p84 mouse monoclonal (Santa Cruz; CA, USA; Western Blot 1∶1000), anti-transferrin receptor mouse monoclonal (Santa Cruz, USA; Wesern Blot 1∶1000).

Techniques: Membrane, Western Blot