gp96MD expression is restricted to mouse basophils.

<p><i>A</i>, Murine mast cells express intact gp96 without evidence for gp96MD. Left panel showed flow profile of bone marrow-derived mast cells. Right panel demonstrates gp96 immunoblot. <i>B</i>, Purified human basophils (left panel) only express full-length gp96 (right panel). <i>C</i>, Human KU812 basophil cell line does not cleave gp96 with and without ER stress induced by tunicamycin (Tu) or thapsigargin (Thap), demonstrated by gp96 immunoblot. <i>D</i>, Western blots for gp96 in the whole cell lysate of KU812 cells with and without ectopic expression of mMCP11.</p

Expression of gp96MD is mediated by proteolysis.

<p><i>A</i>, BMDBs were treated with AEBSF for 6 hours followed by intracellular stain for gp96 using either C-terminal or N-terminal specific antibody. <i>B</i>, BMDBs were treated with AEBSF for 6 hours followed by subjecting total cell lysate to electrophoresis and immunoblot for gp96. <i>C</i>, Kinetic emergence of intracellular gp96 C-terminal antibody reactivity after treatment of B220⁻IgG⁺ population with AEBSF for the indicated time. Number in the quadrant indicates mean fluorescence intensity of gp96 stain. For comparison, peak intensity of the stain at 6 hours were indicated with a vertical line.</p

Expression of gp96MD is not due to a basophil-specific alternative splicing.

<p>Exon (Ex)-specific PCR analysis of gp96 cDNA from sorted populations of bone marrow cells. (Ex2-3, Ex3-5, Ex3-8, et al. indicates the amplified fragments between different exons; NS: non-specific band). Population I: B220⁻IgG^high, Population II: B220⁺IgG^high, Population III: B220⁺IgG⁻.</p

gp96 deletion does not compromise the global secretory machinery of basophils.

<p><i>A</i>, gp96 immunoblot to demonstrate the efficient knockdown of gp96 by shRNA. <i>B</i>, Efficient cytokine and chemokine production by both <i>WT</i> and gp96 knockdown basophils in response to ionomycin. <i>C</i>, Immunoblot of gp96 after treatment with ionomycin.</p

C-terminal deletion mutants of gp96 are unable to chaperone TLRs and integrins.

<p><i>A</i>, gp96 null preB cells were transduced with retroviral expression vectors for empty vector (EV), full-length gp96 (FL) or gp96 deletion mutants (N355, N603), followed by Western blot with gp96N Ab or β–actin as a loading control. <i>B</i>, Various transfectants in (A) were analyzed by flow cytometry for cell surface expression of gp96 clientele TLR2 and α4 integrin (open histogram). Shaded histogram depicts isotype control.</p

Basophil does not express full-length gp96.

<p><i>A</i>, Intracellular stain of <i>WT</i> and <i>KO</i> B cells with isotype control (shaded histogram) or antibody against gp96N and gp96C (open histogram). <i>B</i>, Basophil (population I) does not express gp96C (open histogram). Shaded histograms are staining result with isotype control antibody. <i>C</i>, Western blot for gp96N and gp96C of sorted populations. β–actin was blotted to indicate equal loading of cell lysates.</p

Bone marrow basophils are marked by strong unfolded protein response.

<p><i>A</i>, Western blot for grp78 and CRT of sorted populations. <i>B</i>, RT-PCR of XBP-1 and its spliced form XBP-1s.</p

Bone marrow B220⁻IgG^high cells are basophils.

<p><i>A</i>, Phenotype of B220⁻IgG^high cells. Numbers represent percentages of gated population or corresponding quadrants. <i>B</i>, Phenotypic analysis of FACS sorted BM cells based on cell surface markers of B220 and IgG. (C) RT-PCR analysis of IL-4 and Igα mRNA.</p

Basophil develops in the absence of gp96.

<p><i>A</i>, Presence of B220⁻IgG^high basophil population (boxed) in the bone marrow of gp96 <i>KO</i> mice. <i>B</i>, Intracellular stain demonstrated an efficient deletion of gp96 in bone marrow cells of <i>Hsp90b1^floxRosa26^ERcre</i> (<i>KO</i>) mice by TAM. <i>C</i>, Both <i>WT</i> and gp96 <i>KO</i> B220⁻IgG^high basophils express FcεRI (open histogram indicates gp96 or FcεRI; shaded histogram, isotype control).</p

Correlations between the clinicopathologic characteristics and dyskerin protein expression in hepatocellular carcinoma.

<p>AFP, alpha-fetoprotein; HbsAg, hepatitis B surface antigen.</p>*<p>: Two-tailed chi-Square test. 0.00 indicates <0.01.</p><p>&: Tumor size was measured based on the length of the largest tumor nodule.</p>§<p>: Some data were not available, and the statistical analysis was based on the available data.</p