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Proteintech rabbit antibodies against hspa13
<t> Hspa13 </t> mRNA was increased in atacicept-induced plasmablasts (PBs) and plasma cells (PCs).

Rabbit Antibodies Against Hspa13, supplied by Proteintech, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 92 stars, based on 8 article reviews

rabbit antibodies against hspa13 - by Bioz Stars, 2026-06

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1) Product Images from "Hspa13 Promotes Plasma Cell Production and Antibody Secretion"

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2020.00913

Figure Legend Snippet: Hspa13 mRNA was increased in atacicept-induced plasmablasts (PBs) and plasma cells (PCs).

Techniques Used: Clinical Proteomics, Marker

Figure Legend Snippet: Hspa13 mRNA was increased in LPS-induced PBs/PCs.

Techniques Used: Microarray, Marker

Hspa13 was highly expressed in plasmablasts (PBs) and plasma cells (PCs). (A–C) Hspa13 expression was up-regulated in LPS-induced PBs/PCs. The splenocytes were separated from six 7- to 9-week-old C57BL/6 mice. B cells were sorted by B220 microbeads and stimulated for 0, 24, 48, and 72 hours (hrs) in vitro by 10 μg/ml LPS. Cells were collected and subjected to qPCR (A) , RT-PCR/agarose (B) , and western blot (C) analysis. DNA band ( B , upper panel) was verified as Hspa13 by DNA sequencing. (D,E) Hspa13 was highly expressed in PBs and PCs but not in naïve B cells or germinal center (GC) B cells. Lymphocytes were collected from splenocytes and lymph nodes (LNs) of six sheep red cell (SRC)-immunizated C57BL/6 mice described in . Naïve B cells (CD19 + B220 + IgM + IgD + ), GC B cells (CD19 + B220 + GL7 + CD38 low ), PBs (TACI + CD138 + B220 int CD19 int ), and PCs (TACI + CD138 + B220 − CD19 − ) were sorted by flow cytometry (FACS) and described in , and subjected to qPCR (D) and western blot (E) analysis. (A–E) Data represent three independent experiments with six individual mice each. (A,D) Data were analyzed by the one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). ** P < 0.01, *** P < 0.001.

Figure Legend Snippet: Hspa13 was highly expressed in plasmablasts (PBs) and plasma cells (PCs). (A–C) Hspa13 expression was up-regulated in LPS-induced PBs/PCs. The splenocytes were separated from six 7- to 9-week-old C57BL/6 mice. B cells were sorted by B220 microbeads and stimulated for 0, 24, 48, and 72 hours (hrs) in vitro by 10 μg/ml LPS. Cells were collected and subjected to qPCR (A) , RT-PCR/agarose (B) , and western blot (C) analysis. DNA band ( B , upper panel) was verified as Hspa13 by DNA sequencing. (D,E) Hspa13 was highly expressed in PBs and PCs but not in naïve B cells or germinal center (GC) B cells. Lymphocytes were collected from splenocytes and lymph nodes (LNs) of six sheep red cell (SRC)-immunizated C57BL/6 mice described in . Naïve B cells (CD19 + B220 + IgM + IgD + ), GC B cells (CD19 + B220 + GL7 + CD38 low ), PBs (TACI + CD138 + B220 int CD19 int ), and PCs (TACI + CD138 + B220 − CD19 − ) were sorted by flow cytometry (FACS) and described in , and subjected to qPCR (D) and western blot (E) analysis. (A–E) Data represent three independent experiments with six individual mice each. (A,D) Data were analyzed by the one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). ** P < 0.01, *** P < 0.001.

Techniques Used: Clinical Proteomics, Expressing, In Vitro, Reverse Transcription Polymerase Chain Reaction, Western Blot, DNA Sequencing, Flow Cytometry

Figure Legend Snippet: Hspa13 mRNA was expressed in PBs.

Techniques Used: Expressing

CD19 cre Hspa13 fl/fl mice were developed. (A) A construction map of Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice. This project used the principle of homologous recombination and adopted embryonic stem (ES) cell targeting to modify the Hspa13 locus (Chr16:75755190-75767276 bp) by flox modification. The brief process is as follows: The BAC clone containing the gene of interest was purchased from the Sanger Institute (UK). The ES cell targeting vector was constructed by the ET-clone method. The vector contains a 3.4 Kb 5′ homology arm, a 541 bp flox region, and a PGK-neo-polyA, 3.6 kb 3′ homology arm, plus MC1-TK-polyA negative selection marker. After the vector was linearized, JM8A3 ES cells were transfected electrically. A total of 96 resistant clones were obtained after screening with the G418 and Ganc drugs. A total of 13 positive clones with correct homologous recombination were identified by long fragment PCR. Positive ES cell clones were expanded and injected into blastocysts of C57BL/6J mice to obtain chimeric mice. A high proportion of chimeric mice were mated with C57BL/6J mice to obtain seven positive F1 mice. Hspa13 gene flox heterozygous mice showed no significant abnormalities. After mating the flox mouse with a heterologous CD19 cre mouse, the progeny of the flox homozygous, Cre-positive mouse was knocked out, resulting in a functional loss of the gene of interest in B cells. (B) Hspa13 was knocked out in PBs/PCs from CD19 cre Hspa13 fl/fl mice. PCs (TACI + CD138 + B220 − CD19 − ) were sorted from the spleens and bone marrows (BMs) of 7- to 9-week-old heterologous CD19 cre , Hspa13 fl/fl , and CD19 cre Hspa13 fl/fl mice by FACS. PCs were subjected to PCR (B) and western blot (C) analysis. PCR products: with cre activity: 1,468 bp; with no cre activity: 2,120 bp; wild type: 1,897 bp. (B,C) Data represent three independent experiments with three individual mice each.

Figure Legend Snippet: CD19 cre Hspa13 fl/fl mice were developed. (A) A construction map of Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice. This project used the principle of homologous recombination and adopted embryonic stem (ES) cell targeting to modify the Hspa13 locus (Chr16:75755190-75767276 bp) by flox modification. The brief process is as follows: The BAC clone containing the gene of interest was purchased from the Sanger Institute (UK). The ES cell targeting vector was constructed by the ET-clone method. The vector contains a 3.4 Kb 5′ homology arm, a 541 bp flox region, and a PGK-neo-polyA, 3.6 kb 3′ homology arm, plus MC1-TK-polyA negative selection marker. After the vector was linearized, JM8A3 ES cells were transfected electrically. A total of 96 resistant clones were obtained after screening with the G418 and Ganc drugs. A total of 13 positive clones with correct homologous recombination were identified by long fragment PCR. Positive ES cell clones were expanded and injected into blastocysts of C57BL/6J mice to obtain chimeric mice. A high proportion of chimeric mice were mated with C57BL/6J mice to obtain seven positive F1 mice. Hspa13 gene flox heterozygous mice showed no significant abnormalities. After mating the flox mouse with a heterologous CD19 cre mouse, the progeny of the flox homozygous, Cre-positive mouse was knocked out, resulting in a functional loss of the gene of interest in B cells. (B) Hspa13 was knocked out in PBs/PCs from CD19 cre Hspa13 fl/fl mice. PCs (TACI + CD138 + B220 − CD19 − ) were sorted from the spleens and bone marrows (BMs) of 7- to 9-week-old heterologous CD19 cre , Hspa13 fl/fl , and CD19 cre Hspa13 fl/fl mice by FACS. PCs were subjected to PCR (B) and western blot (C) analysis. PCR products: with cre activity: 1,468 bp; with no cre activity: 2,120 bp; wild type: 1,897 bp. (B,C) Data represent three independent experiments with three individual mice each.

Techniques Used: Homologous Recombination, Modification, Plasmid Preparation, Construct, Selection, Marker, Transfection, Clone Assay, Injection, Functional Assay, Western Blot, Activity Assay

PBs, PCs, and antibodies were reduced in CD19 cre Hspa13 fl/fl (Hspa13 cKO) mice. (A) Hspa13 cKO did not affect naïve B cells or germinal center (GC) B cells in mice. Splenic lymphocytes from 9-week-old wild type, Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were separated using a lymphocyte separation solution; stained with isotype control antibodies, anti-mouse CD19, B220, CD38, and GL7 antibodies; and then analyzed by FACS. The percentages (left panel) and the absolute numbers (right panel) of CD19 + B220 + B cells and CD38 lo GL7 hi B220 + CD19 + GC B cells are shown. (B) Hspa13 cKO reduced PBs, early PCs, and mature PCs in mice. Lymphocytes from the spleen, LNs, and BMs of 9-week-old WT, Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were separated using a lymphocyte separation solution; stained with isotype control antibodies, anti-mouse TACI, CD19, B220, and CD138 antibodies; and then analyzed by FACS. The percentages (left panel) and the absolute numbers (right panel) of TACI + CD138 + B220 int CD19 int PBs, TACI + CD138 + B220 − CD19 int early PCs, and TACI + CD138 + B220 − CD19 − mature PCs are shown. (C) Hspa13 cKO reduced antibodies in mice. Sera were collected from 9-week-old WT, Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice, and the total IgM, IgG, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibody levels were analyzed by ELISA. (A–C) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by the one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure Legend Snippet: PBs, PCs, and antibodies were reduced in CD19 cre Hspa13 fl/fl (Hspa13 cKO) mice. (A) Hspa13 cKO did not affect naïve B cells or germinal center (GC) B cells in mice. Splenic lymphocytes from 9-week-old wild type, Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were separated using a lymphocyte separation solution; stained with isotype control antibodies, anti-mouse CD19, B220, CD38, and GL7 antibodies; and then analyzed by FACS. The percentages (left panel) and the absolute numbers (right panel) of CD19 + B220 + B cells and CD38 lo GL7 hi B220 + CD19 + GC B cells are shown. (B) Hspa13 cKO reduced PBs, early PCs, and mature PCs in mice. Lymphocytes from the spleen, LNs, and BMs of 9-week-old WT, Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were separated using a lymphocyte separation solution; stained with isotype control antibodies, anti-mouse TACI, CD19, B220, and CD138 antibodies; and then analyzed by FACS. The percentages (left panel) and the absolute numbers (right panel) of TACI + CD138 + B220 int CD19 int PBs, TACI + CD138 + B220 − CD19 int early PCs, and TACI + CD138 + B220 − CD19 − mature PCs are shown. (C) Hspa13 cKO reduced antibodies in mice. Sera were collected from 9-week-old WT, Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice, and the total IgM, IgG, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibody levels were analyzed by ELISA. (A–C) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by the one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). * P < 0.05, ** P < 0.01, *** P < 0.001.

Techniques Used: Staining, Control, Enzyme-linked Immunosorbent Assay

Hspa13 cKO reduced the production of LPS-induced PBs, PCs, and antibodies. Splenic B cells from 9-week-old Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were sorted with B220 microbeads and were then stimulated with 10 μg/ml LPS for 0, 1, 2, and 3 days. (A) Hspa13 cKO did not affect LPS-stimulated B-cell proliferation. On days 0, 1, 2, and 3 following LPS stimulation, a CCK8 assay was used to evaluate the cell proliferation. (B,C) Hspa13 cKO did not affect LPS-stimulated B-cell activation. On day 3 following LPS stimulation, cells were stained with isotype control antibodies, anti-mouse B220 and GL7 antibodies, and analyzed by FACS. The percentages (B) and the absolute numbers (C) of B220 + GL7 + B cells are shown. (D,E) Hspa13 cKO reduced LPS-induced PBs, early PCs, and mature PCs. On day 3 following LPS stimulation, cells were stained with isotype control antibodies, anti-mouse TACI, CD19, B220, and CD138 antibodies, and were then analyzed by FACS. The percentages (D) and the absolute numbers (E) of TACI + CD138 + B220 int CD19 int PBs, TACI + CD138 + B220 − CD19 int early PCs, and TACI + CD138 + B220 − CD19 − mature PCs are shown. (F) Hspa13 cKO reduced LPS-induced antibody secretion. On day 3 following LPS stimulation, culture supernatants were collected and the total IgM, IgG1, IgG2b, IgG2c, and IgG3 antibody levels were analyzed by ELISA. (A–F) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by two-way (A) and one-way (C,E,F) ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). *** P < 0.001.

Figure Legend Snippet: Hspa13 cKO reduced the production of LPS-induced PBs, PCs, and antibodies. Splenic B cells from 9-week-old Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were sorted with B220 microbeads and were then stimulated with 10 μg/ml LPS for 0, 1, 2, and 3 days. (A) Hspa13 cKO did not affect LPS-stimulated B-cell proliferation. On days 0, 1, 2, and 3 following LPS stimulation, a CCK8 assay was used to evaluate the cell proliferation. (B,C) Hspa13 cKO did not affect LPS-stimulated B-cell activation. On day 3 following LPS stimulation, cells were stained with isotype control antibodies, anti-mouse B220 and GL7 antibodies, and analyzed by FACS. The percentages (B) and the absolute numbers (C) of B220 + GL7 + B cells are shown. (D,E) Hspa13 cKO reduced LPS-induced PBs, early PCs, and mature PCs. On day 3 following LPS stimulation, cells were stained with isotype control antibodies, anti-mouse TACI, CD19, B220, and CD138 antibodies, and were then analyzed by FACS. The percentages (D) and the absolute numbers (E) of TACI + CD138 + B220 int CD19 int PBs, TACI + CD138 + B220 − CD19 int early PCs, and TACI + CD138 + B220 − CD19 − mature PCs are shown. (F) Hspa13 cKO reduced LPS-induced antibody secretion. On day 3 following LPS stimulation, culture supernatants were collected and the total IgM, IgG1, IgG2b, IgG2c, and IgG3 antibody levels were analyzed by ELISA. (A–F) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by two-way (A) and one-way (C,E,F) ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). *** P < 0.001.

Techniques Used: CCK-8 Assay, Activation Assay, Staining, Control, Enzyme-linked Immunosorbent Assay

Figure Legend Snippet: Hspa13 deficiency reduced Prdm1 and Xbp1 expression.

Techniques Used: Expressing, Marker

Hspa13 cKO reduced sheep red blood cell (SRC)-induced PB/PC and antibody production. Nine-week-old female Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were injected intraperitoneally (i.p.) with 1 × 10 9 SRCs on days 0 and 7. (A,B) Hspa13 cKO did not affect SRC-induce GC B-cell production. On day 21 following SRC stimulation, splenic lymphocytes were stained with isotype control antibodies, anti-mouse CD19, B220, CD38, and GL7 antibodies, and were then analyzed by FACS. The percentages (A) and the absolute numbers (B) of CD38 lo GL7 hi GC cells gated on CD19 + B220 + are shown. (C,D) Hspa13 cKO did not affect the SRC-induced dark zone (DZ) and light zone (LZ) GC B-cell production. On day 21 following SRC stimulation, splenic lymphocytes were stained with anti-mouse CD19, B220, CD38, GL7, CXCR4, and CD86 antibodies, and were then analyzed by FACS. The percentages (C) and the absolute numbers (D) of CXCR4 hi CD86 lo DZ and CXCR4 lo CD86 hi LZ GC B cells gated on CD19 + B220 + CD38 lo GL7 hi GC cells are shown. (E,F) Hspa13 cKO reduced SRC-induced IgG1-, IgG2b-, IgG2c-, and IgG3-expressing PBs/PCs. On day 21 following SRC stimulation, splenic lymphocytes were collected and intracellular staining was performed with isotype control antibodies, anti-mouse B220, IgG1, IgG2b, IgG2c, and IgG3 antibodies. The percentages (E) and the absolute numbers (F) of IgG1-, IgG2b-, IgG2c-, and IgG3-expressing B220 + PBs and B220 − PCs are shown. (G) Hspa13 cKO reduced SRC-induced antibody secretion. On day 21 following SRC stimulation, sera were collected and the total IgM, IgG, IgG1, IgG2b, IgG2c, and IgG3 antibody levels were analyzed by ELISA. (A–G) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by two-way (D) and one-way (B,F,G) ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). ** P < 0.01, *** P < 0.001.

Figure Legend Snippet: Hspa13 cKO reduced sheep red blood cell (SRC)-induced PB/PC and antibody production. Nine-week-old female Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were injected intraperitoneally (i.p.) with 1 × 10 9 SRCs on days 0 and 7. (A,B) Hspa13 cKO did not affect SRC-induce GC B-cell production. On day 21 following SRC stimulation, splenic lymphocytes were stained with isotype control antibodies, anti-mouse CD19, B220, CD38, and GL7 antibodies, and were then analyzed by FACS. The percentages (A) and the absolute numbers (B) of CD38 lo GL7 hi GC cells gated on CD19 + B220 + are shown. (C,D) Hspa13 cKO did not affect the SRC-induced dark zone (DZ) and light zone (LZ) GC B-cell production. On day 21 following SRC stimulation, splenic lymphocytes were stained with anti-mouse CD19, B220, CD38, GL7, CXCR4, and CD86 antibodies, and were then analyzed by FACS. The percentages (C) and the absolute numbers (D) of CXCR4 hi CD86 lo DZ and CXCR4 lo CD86 hi LZ GC B cells gated on CD19 + B220 + CD38 lo GL7 hi GC cells are shown. (E,F) Hspa13 cKO reduced SRC-induced IgG1-, IgG2b-, IgG2c-, and IgG3-expressing PBs/PCs. On day 21 following SRC stimulation, splenic lymphocytes were collected and intracellular staining was performed with isotype control antibodies, anti-mouse B220, IgG1, IgG2b, IgG2c, and IgG3 antibodies. The percentages (E) and the absolute numbers (F) of IgG1-, IgG2b-, IgG2c-, and IgG3-expressing B220 + PBs and B220 − PCs are shown. (G) Hspa13 cKO reduced SRC-induced antibody secretion. On day 21 following SRC stimulation, sera were collected and the total IgM, IgG, IgG1, IgG2b, IgG2c, and IgG3 antibody levels were analyzed by ELISA. (A–G) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by two-way (D) and one-way (B,F,G) ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). ** P < 0.01, *** P < 0.001.

Techniques Used: Injection, Staining, Control, Expressing, Enzyme-linked Immunosorbent Assay

Hspa13 cKO reduced 4-hydroxy-3-nitrophenylacetyl (NP)-specific antibody production. Nine-week-old Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were injected i.p. with T cell-independent antigen NP-Ficoll (A) and T cell-dependent antigen NP-keyhole lymphocyte hemocyanin (KLH) (B) on days 0 and 7. On day 21 following NP-Ficoll (A) or NP-KLH (B) stimulation, sera were collected and the NP-specific IgM, IgG, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibody levels were analyzed by ELISA. (A,B) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure Legend Snippet: Hspa13 cKO reduced 4-hydroxy-3-nitrophenylacetyl (NP)-specific antibody production. Nine-week-old Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were injected i.p. with T cell-independent antigen NP-Ficoll (A) and T cell-dependent antigen NP-keyhole lymphocyte hemocyanin (KLH) (B) on days 0 and 7. On day 21 following NP-Ficoll (A) or NP-KLH (B) stimulation, sera were collected and the NP-specific IgM, IgG, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibody levels were analyzed by ELISA. (A,B) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). * P < 0.05, ** P < 0.01, *** P < 0.001.

Techniques Used: Injection, Enzyme-linked Immunosorbent Assay

Hspa13 cKO reduced class switch recombination (CSR), somatic hypermutation (SHM), and affinity maturation of antibodies. Nine-week-old female Hspa13 fl/fl (control) and CD19 cre Hspa13 fl/fl (Hspa13 cKO) mice (three mice per group) were injected i.p. with 1 × 10 9 SRCs (A–C) or NP-KLH (D,E) on days 0 and 7. On day 21 following SRC stimulation, splenocytes were stained with PerCP-conjugated anti-mouse B220 antibodies and sorted by FACS. Single cells were captured using the 10 X Genomics Full Chromium platform and subjected to RNA- and VDJ-sequencing. (A) Hspa13 cKO reduced SRC-induced PBs. Of the single PBs, 27 (3.49%) and 11 (1.07%) (Ighm + , Ighg1 + , Ighg2b + , Ighg2c + , Ighg3 + , Igha + , or Ighe + Cd3d − Cd3e − Cd3gCd4 − Cd8a − Cd19 + Ptprc + Ms4a1 + Ighd − Bcl6 − Aicda − Prdm1 + Xbp1 + Sdc1 + ) within the splenic B cell population were identified by single-cell RNA-sequencing out of 774 and 1,025 single cells corresponding to CD19 cre Hspa13 fl/fl and Hspa13 fl/fl mice, respectively. (B) Hspa13 cKO reduced SRC-induced antibody CSR. Single cells expressing genes encoding IgD, IgM, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibodies were identified by single-cell VDJ-sequencing. The percentage of different antibody subtypes expressed by single cells out of 734 and 382 antibody-expressing single cells from CD19 cre Hspa13 fl/fl and Hspa13 fl/fl mice, respectively, is shown. (C) Hspa13 cKO reduced SRC-induced antibody SHM. The single antibody gene was determined by single-cell VDJ-sequencing. SHM percentages in the CDR (complementarity-determining region) of the heavy (H) and light (L) chains are based on 382 and 734 antibody genes from Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice, respectively. (D) Hspa13 cKO reduced NP-specific SHM induced by NP-KLH. The distribution of the number of mutations per unique clone (VH186.2 segment) is shown. Numbers refer to 100 individual sequences; three animals per group were analyzed. (E) Hspa13 cKO reduced NP-specific high-affinity clones induced by NP-KLH. On day 21 following NP-KLH stimulation, the percentage of NP high-affinity clones containing the W33L mutation in CDR1 in purified GC B cells of Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice was determined. Each dot corresponds to a single animal (30 unique clones/mouse; Mann-Whitney test; error bars represent s.e.m; *** P < 0.001).

Figure Legend Snippet: Hspa13 cKO reduced class switch recombination (CSR), somatic hypermutation (SHM), and affinity maturation of antibodies. Nine-week-old female Hspa13 fl/fl (control) and CD19 cre Hspa13 fl/fl (Hspa13 cKO) mice (three mice per group) were injected i.p. with 1 × 10 9 SRCs (A–C) or NP-KLH (D,E) on days 0 and 7. On day 21 following SRC stimulation, splenocytes were stained with PerCP-conjugated anti-mouse B220 antibodies and sorted by FACS. Single cells were captured using the 10 X Genomics Full Chromium platform and subjected to RNA- and VDJ-sequencing. (A) Hspa13 cKO reduced SRC-induced PBs. Of the single PBs, 27 (3.49%) and 11 (1.07%) (Ighm + , Ighg1 + , Ighg2b + , Ighg2c + , Ighg3 + , Igha + , or Ighe + Cd3d − Cd3e − Cd3gCd4 − Cd8a − Cd19 + Ptprc + Ms4a1 + Ighd − Bcl6 − Aicda − Prdm1 + Xbp1 + Sdc1 + ) within the splenic B cell population were identified by single-cell RNA-sequencing out of 774 and 1,025 single cells corresponding to CD19 cre Hspa13 fl/fl and Hspa13 fl/fl mice, respectively. (B) Hspa13 cKO reduced SRC-induced antibody CSR. Single cells expressing genes encoding IgD, IgM, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibodies were identified by single-cell VDJ-sequencing. The percentage of different antibody subtypes expressed by single cells out of 734 and 382 antibody-expressing single cells from CD19 cre Hspa13 fl/fl and Hspa13 fl/fl mice, respectively, is shown. (C) Hspa13 cKO reduced SRC-induced antibody SHM. The single antibody gene was determined by single-cell VDJ-sequencing. SHM percentages in the CDR (complementarity-determining region) of the heavy (H) and light (L) chains are based on 382 and 734 antibody genes from Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice, respectively. (D) Hspa13 cKO reduced NP-specific SHM induced by NP-KLH. The distribution of the number of mutations per unique clone (VH186.2 segment) is shown. Numbers refer to 100 individual sequences; three animals per group were analyzed. (E) Hspa13 cKO reduced NP-specific high-affinity clones induced by NP-KLH. On day 21 following NP-KLH stimulation, the percentage of NP high-affinity clones containing the W33L mutation in CDR1 in purified GC B cells of Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice was determined. Each dot corresponds to a single animal (30 unique clones/mouse; Mann-Whitney test; error bars represent s.e.m; *** P < 0.001).

Techniques Used: Control, Injection, Staining, Sequencing, RNA Sequencing, Expressing, Clone Assay, Mutagenesis, Purification, MANN-WHITNEY

Hspa13 interacts with endoplasmic reticulum (ER) proteins involved in positive regulation of protein transport from the ER to the cytosol. Splenic B220 + B cells from 9-week-old C57BL/6 mice (three mice per group) were sorted using B220 microbeads and were then stimulated for 3 days with 10 μg/ml LPS. LPS-stimulated B cells were collected for anti-Hspa13 antibody co-immunoprecipitation (IP) experiments. Co-immunoprecipitated proteins were identified by mass spectrometry. (A) SDS-PAGE and silver staining results showing affinity captured interacting proteins from whole cell extracts. Putative interacting protein bands marked as 1, 2, 3, and 4 were excised for mass spectrometry analysis. (B) Anti-Hspa13 antibody specifically co-immunoprecipitated 56 proteins. We identified 393, 56, and 148 proteins that were co-immunoprecipitated by control IgG antibodies only, anti-Hspa13 antibodies only, and the two antibodies together, respectively. (C) Bar plot ranking of the top 10 cellular components (CC), based on enrichment score. Gene ontology (GO)-analysis was performed using a gene ontology website ( http://www.geneontology.org/ ). (D) Bar plot ranking of the top 10 biologic processes (BP) based on enrichment score. GO-analysis was performed based on the gene ontology website ( http://www.geneontology.org/ ). (E) A list of the 10 best hits of Hspa13 interacting partners. Detailed interacting protein data are shown in . (F) Interaction of Hspa13 and Bcap31. The recombinant plasmids expressing Hspa13-V5 and Bcap31-Flag were transiently transfected into 293T cells. At 48 hrs after transfection, cells were lysed and anti-V5 antibody was used to immunoprecipitate proteins probed with anti-Flag antibody. Data are shown for one representative experiment from three independent experiments with similar results. (G) Hspa13 cKO reduced Bcap31 mRNA expression in PBs induced by SRC. Bcap31 mRNA expression was analyzed from 10 and 8 PBs from the single-cell RNA sequencing data of SRC-primed Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice, respectively, described in . Student's t -test (two tailed). Error bars represent s.e.m. * P < 0.05.

Figure Legend Snippet: Hspa13 interacts with endoplasmic reticulum (ER) proteins involved in positive regulation of protein transport from the ER to the cytosol. Splenic B220 + B cells from 9-week-old C57BL/6 mice (three mice per group) were sorted using B220 microbeads and were then stimulated for 3 days with 10 μg/ml LPS. LPS-stimulated B cells were collected for anti-Hspa13 antibody co-immunoprecipitation (IP) experiments. Co-immunoprecipitated proteins were identified by mass spectrometry. (A) SDS-PAGE and silver staining results showing affinity captured interacting proteins from whole cell extracts. Putative interacting protein bands marked as 1, 2, 3, and 4 were excised for mass spectrometry analysis. (B) Anti-Hspa13 antibody specifically co-immunoprecipitated 56 proteins. We identified 393, 56, and 148 proteins that were co-immunoprecipitated by control IgG antibodies only, anti-Hspa13 antibodies only, and the two antibodies together, respectively. (C) Bar plot ranking of the top 10 cellular components (CC), based on enrichment score. Gene ontology (GO)-analysis was performed using a gene ontology website ( http://www.geneontology.org/ ). (D) Bar plot ranking of the top 10 biologic processes (BP) based on enrichment score. GO-analysis was performed based on the gene ontology website ( http://www.geneontology.org/ ). (E) A list of the 10 best hits of Hspa13 interacting partners. Detailed interacting protein data are shown in . (F) Interaction of Hspa13 and Bcap31. The recombinant plasmids expressing Hspa13-V5 and Bcap31-Flag were transiently transfected into 293T cells. At 48 hrs after transfection, cells were lysed and anti-V5 antibody was used to immunoprecipitate proteins probed with anti-Flag antibody. Data are shown for one representative experiment from three independent experiments with similar results. (G) Hspa13 cKO reduced Bcap31 mRNA expression in PBs induced by SRC. Bcap31 mRNA expression was analyzed from 10 and 8 PBs from the single-cell RNA sequencing data of SRC-primed Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice, respectively, described in . Student's t -test (two tailed). Error bars represent s.e.m. * P < 0.05.

Techniques Used: Immunoprecipitation, Mass Spectrometry, SDS Page, Silver Staining, Control, Recombinant, Expressing, Transfection, RNA Sequencing, Two Tailed Test

Figure Legend Snippet: Hspa13 mRNA was increased in B220 + cells from patients with multiple myeloma (MM).

Techniques Used: Marker

Figure Legend Snippet: Hspa13 mRNA was increased in B220 + cells from patients with systemic lupus erythematosus (SLE).

Techniques Used: Marker

Hspa13 cKO reduced autoantibodies and proteinuria in pristane-induced lupus and lupus-prone MRL/lpr mouse model. (A) Hspa13 cKO reduced autoantibodies in pristane-induced lupus mice. To induce lupus, 9-week-old female Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice on a B6 background were injected i.p. with 0.5 ml of pristane. On day 21 following pristane stimulation, sera were collected and dsDNA-specific IgM and IgG antibody levels were analyzed by ELISA. (B) Hspa13 cKO reduced proteinuria in the pristane-induced lupus mice. On day 21 following pristane stimulation, urine was collected and proteinuria was measured. (C) Hspa13 cKO reduced autoantibodies in a lupus-prone MRL/lpr mouse model. Sera were collected from Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice in lupus-prone MRL/lpr mice background at 6 months of age and dsDNA-specific IgM and IgG antibody levels were analyzed by ELISA. (D) Hspa13 cKO reduced proteinuria in the lupus-prone MRL/lpr mouse model. Urine was collected from Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice in a lupus-prone MRL/lpr mice background at 6 months of age and proteinuria was measured. (A–D) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by the one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). ** P < 0.01, *** P < 0.01.

Figure Legend Snippet: Hspa13 cKO reduced autoantibodies and proteinuria in pristane-induced lupus and lupus-prone MRL/lpr mouse model. (A) Hspa13 cKO reduced autoantibodies in pristane-induced lupus mice. To induce lupus, 9-week-old female Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice on a B6 background were injected i.p. with 0.5 ml of pristane. On day 21 following pristane stimulation, sera were collected and dsDNA-specific IgM and IgG antibody levels were analyzed by ELISA. (B) Hspa13 cKO reduced proteinuria in the pristane-induced lupus mice. On day 21 following pristane stimulation, urine was collected and proteinuria was measured. (C) Hspa13 cKO reduced autoantibodies in a lupus-prone MRL/lpr mouse model. Sera were collected from Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice in lupus-prone MRL/lpr mice background at 6 months of age and dsDNA-specific IgM and IgG antibody levels were analyzed by ELISA. (D) Hspa13 cKO reduced proteinuria in the lupus-prone MRL/lpr mouse model. Urine was collected from Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice in a lupus-prone MRL/lpr mice background at 6 months of age and proteinuria was measured. (A–D) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by the one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). ** P < 0.01, *** P < 0.01.

Techniques Used: Injection, Enzyme-linked Immunosorbent Assay

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Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 mRNA was increased in atacicept-induced plasmablasts (PBs) and plasma cells (PCs).

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Clinical Proteomics, Marker

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 mRNA was increased in LPS-induced PBs/PCs.

Techniques: Microarray, Marker

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 was highly expressed in plasmablasts (PBs) and plasma cells (PCs). (A–C) Hspa13 expression was up-regulated in LPS-induced PBs/PCs. The splenocytes were separated from six 7- to 9-week-old C57BL/6 mice. B cells were sorted by B220 microbeads and stimulated for 0, 24, 48, and 72 hours (hrs) in vitro by 10 μg/ml LPS. Cells were collected and subjected to qPCR (A) , RT-PCR/agarose (B) , and western blot (C) analysis. DNA band ( B , upper panel) was verified as Hspa13 by DNA sequencing. (D,E) Hspa13 was highly expressed in PBs and PCs but not in naïve B cells or germinal center (GC) B cells. Lymphocytes were collected from splenocytes and lymph nodes (LNs) of six sheep red cell (SRC)-immunizated C57BL/6 mice described in . Naïve B cells (CD19 + B220 + IgM + IgD + ), GC B cells (CD19 + B220 + GL7 + CD38 low ), PBs (TACI + CD138 + B220 int CD19 int ), and PCs (TACI + CD138 + B220 − CD19 − ) were sorted by flow cytometry (FACS) and described in , and subjected to qPCR (D) and western blot (E) analysis. (A–E) Data represent three independent experiments with six individual mice each. (A,D) Data were analyzed by the one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). ** P < 0.01, *** P < 0.001.

Techniques: Clinical Proteomics, Expressing, In Vitro, Reverse Transcription Polymerase Chain Reaction, Western Blot, DNA Sequencing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 mRNA was expressed in PBs.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: CD19 cre Hspa13 fl/fl mice were developed. (A) A construction map of Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice. This project used the principle of homologous recombination and adopted embryonic stem (ES) cell targeting to modify the Hspa13 locus (Chr16:75755190-75767276 bp) by flox modification. The brief process is as follows: The BAC clone containing the gene of interest was purchased from the Sanger Institute (UK). The ES cell targeting vector was constructed by the ET-clone method. The vector contains a 3.4 Kb 5′ homology arm, a 541 bp flox region, and a PGK-neo-polyA, 3.6 kb 3′ homology arm, plus MC1-TK-polyA negative selection marker. After the vector was linearized, JM8A3 ES cells were transfected electrically. A total of 96 resistant clones were obtained after screening with the G418 and Ganc drugs. A total of 13 positive clones with correct homologous recombination were identified by long fragment PCR. Positive ES cell clones were expanded and injected into blastocysts of C57BL/6J mice to obtain chimeric mice. A high proportion of chimeric mice were mated with C57BL/6J mice to obtain seven positive F1 mice. Hspa13 gene flox heterozygous mice showed no significant abnormalities. After mating the flox mouse with a heterologous CD19 cre mouse, the progeny of the flox homozygous, Cre-positive mouse was knocked out, resulting in a functional loss of the gene of interest in B cells. (B) Hspa13 was knocked out in PBs/PCs from CD19 cre Hspa13 fl/fl mice. PCs (TACI + CD138 + B220 − CD19 − ) were sorted from the spleens and bone marrows (BMs) of 7- to 9-week-old heterologous CD19 cre , Hspa13 fl/fl , and CD19 cre Hspa13 fl/fl mice by FACS. PCs were subjected to PCR (B) and western blot (C) analysis. PCR products: with cre activity: 1,468 bp; with no cre activity: 2,120 bp; wild type: 1,897 bp. (B,C) Data represent three independent experiments with three individual mice each.

Techniques: Homologous Recombination, Modification, Plasmid Preparation, Construct, Selection, Marker, Transfection, Clone Assay, Injection, Functional Assay, Western Blot, Activity Assay

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: PBs, PCs, and antibodies were reduced in CD19 cre Hspa13 fl/fl (Hspa13 cKO) mice. (A) Hspa13 cKO did not affect naïve B cells or germinal center (GC) B cells in mice. Splenic lymphocytes from 9-week-old wild type, Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were separated using a lymphocyte separation solution; stained with isotype control antibodies, anti-mouse CD19, B220, CD38, and GL7 antibodies; and then analyzed by FACS. The percentages (left panel) and the absolute numbers (right panel) of CD19 + B220 + B cells and CD38 lo GL7 hi B220 + CD19 + GC B cells are shown. (B) Hspa13 cKO reduced PBs, early PCs, and mature PCs in mice. Lymphocytes from the spleen, LNs, and BMs of 9-week-old WT, Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were separated using a lymphocyte separation solution; stained with isotype control antibodies, anti-mouse TACI, CD19, B220, and CD138 antibodies; and then analyzed by FACS. The percentages (left panel) and the absolute numbers (right panel) of TACI + CD138 + B220 int CD19 int PBs, TACI + CD138 + B220 − CD19 int early PCs, and TACI + CD138 + B220 − CD19 − mature PCs are shown. (C) Hspa13 cKO reduced antibodies in mice. Sera were collected from 9-week-old WT, Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice, and the total IgM, IgG, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibody levels were analyzed by ELISA. (A–C) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by the one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). * P < 0.05, ** P < 0.01, *** P < 0.001.

Techniques: Staining, Control, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 cKO reduced the production of LPS-induced PBs, PCs, and antibodies. Splenic B cells from 9-week-old Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were sorted with B220 microbeads and were then stimulated with 10 μg/ml LPS for 0, 1, 2, and 3 days. (A) Hspa13 cKO did not affect LPS-stimulated B-cell proliferation. On days 0, 1, 2, and 3 following LPS stimulation, a CCK8 assay was used to evaluate the cell proliferation. (B,C) Hspa13 cKO did not affect LPS-stimulated B-cell activation. On day 3 following LPS stimulation, cells were stained with isotype control antibodies, anti-mouse B220 and GL7 antibodies, and analyzed by FACS. The percentages (B) and the absolute numbers (C) of B220 + GL7 + B cells are shown. (D,E) Hspa13 cKO reduced LPS-induced PBs, early PCs, and mature PCs. On day 3 following LPS stimulation, cells were stained with isotype control antibodies, anti-mouse TACI, CD19, B220, and CD138 antibodies, and were then analyzed by FACS. The percentages (D) and the absolute numbers (E) of TACI + CD138 + B220 int CD19 int PBs, TACI + CD138 + B220 − CD19 int early PCs, and TACI + CD138 + B220 − CD19 − mature PCs are shown. (F) Hspa13 cKO reduced LPS-induced antibody secretion. On day 3 following LPS stimulation, culture supernatants were collected and the total IgM, IgG1, IgG2b, IgG2c, and IgG3 antibody levels were analyzed by ELISA. (A–F) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by two-way (A) and one-way (C,E,F) ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). *** P < 0.001.

Techniques: CCK-8 Assay, Activation Assay, Staining, Control, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 deficiency reduced Prdm1 and Xbp1 expression.

Techniques: Expressing, Marker

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 cKO reduced sheep red blood cell (SRC)-induced PB/PC and antibody production. Nine-week-old female Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were injected intraperitoneally (i.p.) with 1 × 10 9 SRCs on days 0 and 7. (A,B) Hspa13 cKO did not affect SRC-induce GC B-cell production. On day 21 following SRC stimulation, splenic lymphocytes were stained with isotype control antibodies, anti-mouse CD19, B220, CD38, and GL7 antibodies, and were then analyzed by FACS. The percentages (A) and the absolute numbers (B) of CD38 lo GL7 hi GC cells gated on CD19 + B220 + are shown. (C,D) Hspa13 cKO did not affect the SRC-induced dark zone (DZ) and light zone (LZ) GC B-cell production. On day 21 following SRC stimulation, splenic lymphocytes were stained with anti-mouse CD19, B220, CD38, GL7, CXCR4, and CD86 antibodies, and were then analyzed by FACS. The percentages (C) and the absolute numbers (D) of CXCR4 hi CD86 lo DZ and CXCR4 lo CD86 hi LZ GC B cells gated on CD19 + B220 + CD38 lo GL7 hi GC cells are shown. (E,F) Hspa13 cKO reduced SRC-induced IgG1-, IgG2b-, IgG2c-, and IgG3-expressing PBs/PCs. On day 21 following SRC stimulation, splenic lymphocytes were collected and intracellular staining was performed with isotype control antibodies, anti-mouse B220, IgG1, IgG2b, IgG2c, and IgG3 antibodies. The percentages (E) and the absolute numbers (F) of IgG1-, IgG2b-, IgG2c-, and IgG3-expressing B220 + PBs and B220 − PCs are shown. (G) Hspa13 cKO reduced SRC-induced antibody secretion. On day 21 following SRC stimulation, sera were collected and the total IgM, IgG, IgG1, IgG2b, IgG2c, and IgG3 antibody levels were analyzed by ELISA. (A–G) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by two-way (D) and one-way (B,F,G) ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). ** P < 0.01, *** P < 0.001.

Techniques: Injection, Staining, Control, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 cKO reduced 4-hydroxy-3-nitrophenylacetyl (NP)-specific antibody production. Nine-week-old Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were injected i.p. with T cell-independent antigen NP-Ficoll (A) and T cell-dependent antigen NP-keyhole lymphocyte hemocyanin (KLH) (B) on days 0 and 7. On day 21 following NP-Ficoll (A) or NP-KLH (B) stimulation, sera were collected and the NP-specific IgM, IgG, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibody levels were analyzed by ELISA. (A,B) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). * P < 0.05, ** P < 0.01, *** P < 0.001.

Techniques: Injection, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 cKO reduced class switch recombination (CSR), somatic hypermutation (SHM), and affinity maturation of antibodies. Nine-week-old female Hspa13 fl/fl (control) and CD19 cre Hspa13 fl/fl (Hspa13 cKO) mice (three mice per group) were injected i.p. with 1 × 10 9 SRCs (A–C) or NP-KLH (D,E) on days 0 and 7. On day 21 following SRC stimulation, splenocytes were stained with PerCP-conjugated anti-mouse B220 antibodies and sorted by FACS. Single cells were captured using the 10 X Genomics Full Chromium platform and subjected to RNA- and VDJ-sequencing. (A) Hspa13 cKO reduced SRC-induced PBs. Of the single PBs, 27 (3.49%) and 11 (1.07%) (Ighm + , Ighg1 + , Ighg2b + , Ighg2c + , Ighg3 + , Igha + , or Ighe + Cd3d − Cd3e − Cd3gCd4 − Cd8a − Cd19 + Ptprc + Ms4a1 + Ighd − Bcl6 − Aicda − Prdm1 + Xbp1 + Sdc1 + ) within the splenic B cell population were identified by single-cell RNA-sequencing out of 774 and 1,025 single cells corresponding to CD19 cre Hspa13 fl/fl and Hspa13 fl/fl mice, respectively. (B) Hspa13 cKO reduced SRC-induced antibody CSR. Single cells expressing genes encoding IgD, IgM, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibodies were identified by single-cell VDJ-sequencing. The percentage of different antibody subtypes expressed by single cells out of 734 and 382 antibody-expressing single cells from CD19 cre Hspa13 fl/fl and Hspa13 fl/fl mice, respectively, is shown. (C) Hspa13 cKO reduced SRC-induced antibody SHM. The single antibody gene was determined by single-cell VDJ-sequencing. SHM percentages in the CDR (complementarity-determining region) of the heavy (H) and light (L) chains are based on 382 and 734 antibody genes from Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice, respectively. (D) Hspa13 cKO reduced NP-specific SHM induced by NP-KLH. The distribution of the number of mutations per unique clone (VH186.2 segment) is shown. Numbers refer to 100 individual sequences; three animals per group were analyzed. (E) Hspa13 cKO reduced NP-specific high-affinity clones induced by NP-KLH. On day 21 following NP-KLH stimulation, the percentage of NP high-affinity clones containing the W33L mutation in CDR1 in purified GC B cells of Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice was determined. Each dot corresponds to a single animal (30 unique clones/mouse; Mann-Whitney test; error bars represent s.e.m; *** P < 0.001).

Techniques: Control, Injection, Staining, Sequencing, RNA Sequencing, Expressing, Clone Assay, Mutagenesis, Purification, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 interacts with endoplasmic reticulum (ER) proteins involved in positive regulation of protein transport from the ER to the cytosol. Splenic B220 + B cells from 9-week-old C57BL/6 mice (three mice per group) were sorted using B220 microbeads and were then stimulated for 3 days with 10 μg/ml LPS. LPS-stimulated B cells were collected for anti-Hspa13 antibody co-immunoprecipitation (IP) experiments. Co-immunoprecipitated proteins were identified by mass spectrometry. (A) SDS-PAGE and silver staining results showing affinity captured interacting proteins from whole cell extracts. Putative interacting protein bands marked as 1, 2, 3, and 4 were excised for mass spectrometry analysis. (B) Anti-Hspa13 antibody specifically co-immunoprecipitated 56 proteins. We identified 393, 56, and 148 proteins that were co-immunoprecipitated by control IgG antibodies only, anti-Hspa13 antibodies only, and the two antibodies together, respectively. (C) Bar plot ranking of the top 10 cellular components (CC), based on enrichment score. Gene ontology (GO)-analysis was performed using a gene ontology website ( http://www.geneontology.org/ ). (D) Bar plot ranking of the top 10 biologic processes (BP) based on enrichment score. GO-analysis was performed based on the gene ontology website ( http://www.geneontology.org/ ). (E) A list of the 10 best hits of Hspa13 interacting partners. Detailed interacting protein data are shown in . (F) Interaction of Hspa13 and Bcap31. The recombinant plasmids expressing Hspa13-V5 and Bcap31-Flag were transiently transfected into 293T cells. At 48 hrs after transfection, cells were lysed and anti-V5 antibody was used to immunoprecipitate proteins probed with anti-Flag antibody. Data are shown for one representative experiment from three independent experiments with similar results. (G) Hspa13 cKO reduced Bcap31 mRNA expression in PBs induced by SRC. Bcap31 mRNA expression was analyzed from 10 and 8 PBs from the single-cell RNA sequencing data of SRC-primed Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice, respectively, described in . Student's t -test (two tailed). Error bars represent s.e.m. * P < 0.05.

Techniques: Immunoprecipitation, Mass Spectrometry, SDS Page, Silver Staining, Control, Recombinant, Expressing, Transfection, RNA Sequencing, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 mRNA was increased in B220 + cells from patients with multiple myeloma (MM).

Techniques: Marker

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 mRNA was increased in B220 + cells from patients with systemic lupus erythematosus (SLE).

Techniques: Marker

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 cKO reduced autoantibodies and proteinuria in pristane-induced lupus and lupus-prone MRL/lpr mouse model. (A) Hspa13 cKO reduced autoantibodies in pristane-induced lupus mice. To induce lupus, 9-week-old female Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice on a B6 background were injected i.p. with 0.5 ml of pristane. On day 21 following pristane stimulation, sera were collected and dsDNA-specific IgM and IgG antibody levels were analyzed by ELISA. (B) Hspa13 cKO reduced proteinuria in the pristane-induced lupus mice. On day 21 following pristane stimulation, urine was collected and proteinuria was measured. (C) Hspa13 cKO reduced autoantibodies in a lupus-prone MRL/lpr mouse model. Sera were collected from Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice in lupus-prone MRL/lpr mice background at 6 months of age and dsDNA-specific IgM and IgG antibody levels were analyzed by ELISA. (D) Hspa13 cKO reduced proteinuria in the lupus-prone MRL/lpr mouse model. Urine was collected from Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice in a lupus-prone MRL/lpr mice background at 6 months of age and proteinuria was measured. (A–D) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by the one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). ** P < 0.01, *** P < 0.01.

Techniques: Injection, Enzyme-linked Immunosorbent Assay

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