Journal: Immunology

Article Title: Role of the inflammatory protein serine protease inhibitor Kazal in preventing cytolytic granule granzyme A-mediated apoptosis

doi: 10.1111/j.1365-2567.2011.03498.x

Figure Lengend Snippet: Granzyme A (GzmA) was co-immmunoprecipitated with serine protease inhibitor Kazal (SPIK). The medium from cells expressing high levels of SPIK, such as pancreatic cells, S2–3 cells, and G54 cells, was incubated with 200 ng recombinant GzmA and then incubated at 4° overnight with magnetic beads covalently linked with anti-SPIK monoclonal antibody. SPIK and binding proteins were released from beads by treatment with pH 2·5 buffer and resolved in SDS–PAGE. The proteins were then transferred to the membrane and analysed by Western blot with monoclonal anti-GzmA and anti-SPIK antibodies, respectively. (a) Experimental procedure. (b) Western blots showing SPIK and binding proteins after immune co-precipitation with anti-SPIK antibody. GzmA and SPIK are indicated on blots. (c) Immune co-precipitation with anti-GzmA antibody.

Article Snippet: To induce cell apoptosis, the cells released by digestion with trypsin were incubated with 2 μ m recombinant GzmA and 254 ng/ml perforin (PFR; Abnova, Taipei, Taiwan) at 37° for 20 hr.

Techniques: Protease Inhibitor, Expressing, Incubation, Recombinant, Magnetic Beads, Binding Assay, SDS Page, Membrane, Western Blot

Journal: Immunology

Article Title: Role of the inflammatory protein serine protease inhibitor Kazal in preventing cytolytic granule granzyme A-mediated apoptosis

doi: 10.1111/j.1365-2567.2011.03498.x

Figure Lengend Snippet: Over-expression of serine protease inhibitor Kazal (SPIK) results in cellular resistance to Granzyme A (GzmA) -induced apoptosis. S2–3 and SP23 cells were incubated with GzmA and perforin (PFR), individually or combined. Apoptosis was assessed. (a) Hoechst staining of apoptotic cells. (b) Cell apoptosis was analysed by flow cytometry after staining with annexin V-FITC. (c) Cell apoptosis was quantified by flow cytometry after double staining with propidium iodide (PI) and annexin V-FITC. The lower-right quadrant indicates the apoptotic cells. (d) Percentage of apoptotic cells from flow cytometry with double staining.

Article Snippet: To induce cell apoptosis, the cells released by digestion with trypsin were incubated with 2 μ m recombinant GzmA and 254 ng/ml perforin (PFR; Abnova, Taipei, Taiwan) at 37° for 20 hr.

Techniques: Over Expression, Protease Inhibitor, Incubation, Staining, Flow Cytometry, Double Staining

Journal: Immunology

Article Title: Role of the inflammatory protein serine protease inhibitor Kazal in preventing cytolytic granule granzyme A-mediated apoptosis

doi: 10.1111/j.1365-2567.2011.03498.x

Figure Lengend Snippet: Silencing of over-expressed serine protease inhibitor Kazal (SPIK) in S2–3 cells restores cell sensitivity to Granzyme A (GzmA) -induced cell apoptosis. S2–3 cells were transfected with SPIK small interfering (si) RNA plasmids L71 and L183 or with their vector for mock transfection. The plasmid for woodchuck hepatitis B virus (WHBV) siRNA was used as a control for specificity. SP23 cells, expressing only background levels of SPIK, were used as a control. Cell apoptosis was induced by treatment of cells with GzmA/PFR 3 days after transfection. (a) The SPIK RNA levels were examined by Northern blot with the probe specified for SPIK. (b) SPIK protein was examined by Western blot with monoclonal anti-SPIK antibody. (c) Cell apoptosis was analysed by flow cytometry after staining with annexin V-FITC. (d) Cell apoptosis was quantified by flow cytometry after double staining with propidium iodide (PI) and annexin V-FITC. The lower-right quadrant indicates the apoptotic cells. (e) Percentage of apoptotic cells from flow cytometry with double staining.

Article Snippet: To induce cell apoptosis, the cells released by digestion with trypsin were incubated with 2 μ m recombinant GzmA and 254 ng/ml perforin (PFR; Abnova, Taipei, Taiwan) at 37° for 20 hr.

Techniques: Protease Inhibitor, Transfection, Plasmid Preparation, Virus, Control, Expressing, Northern Blot, Western Blot, Flow Cytometry, Staining, Double Staining

Journal: Immunology

Article Title: Role of the inflammatory protein serine protease inhibitor Kazal in preventing cytolytic granule granzyme A-mediated apoptosis

doi: 10.1111/j.1365-2567.2011.03498.x

Figure Lengend Snippet: The C3–C4 region is critical for the ability of serine protease inhibitor Kazal (SPIK) to suppress Granzyme A (GzmA) -induced apoptosis. Wild-type SPIK plasmid PWT was constructed with pCMVscript vector with a myc tag sequence at the N-terminus. Three mutants PD1, PD2 and PD3 were constructed based on PWT. (a). The structure of PWT and its deletion mutants. (b). HeLa cells were transfected with either wild-type plasmid or its mutants (PD1, PD2 and PD3) or mock transfected with vector as a control (none). Cellular SPIK levels were assessed with Western blot using anti-myc antibody. Actin was used as loading control. (c) Cell apoptosis was analysed by flow cytometry after staining with annexin V-FITC. (d) Cell apoptosis was quantified by flow cytometry after double staining with propidium iodide (PI) and annexin V-FITC. The lower-right quadrant indicates the apoptotic cells. (e) Percentage of apoptotic cells from flow cytometry with double staining.

Article Snippet: To induce cell apoptosis, the cells released by digestion with trypsin were incubated with 2 μ m recombinant GzmA and 254 ng/ml perforin (PFR; Abnova, Taipei, Taiwan) at 37° for 20 hr.

Techniques: Protease Inhibitor, Plasmid Preparation, Construct, Sequencing, Transfection, Control, Western Blot, Flow Cytometry, Staining, Double Staining