antibody targeting spib  (Proteintech)

Journal: Frontiers in Immunology

Article Title: SpiB regulates the expression of B-cell-related genes and increases the longevity of memory B cells

doi: 10.3389/fimmu.2023.1250719

Figure Lengend Snippet: SpiB is expressed in GC B and B mem cells and induced by CD40 stimulation in vitro. (A) SpiB mRNA expression in B cell subsets from mouse spleen collected 14 days post NP-CGG immunization. B cells were sorted as CD19 + Igκ - and NP + CD38 + GL7 - (early B mem cells), NP + CD38 - GL7 + (GC B cells), or CD19 + CD21 lo CD23 hi AA4.1 - [naive follicular B (FoB) cells]. Comparison of early B mem and GC B cells (top) and naïve FoB and GC B cells (bottom) are shown. (B) SpiB mRNA expression in B cells cultured on 40LB feeder cells for 0-3 days (left) and B cells stimulated with the indicated antibodies or LPS (right). (C) Immunofluorescence microscopy of frozen sections of spleens from mice immunized with NP-SRBC 7 days previously. The sections were stained for SpiB (blue), IgD (red), and Thy1.2 (green) (top), or for SpiB (blue) and B220 (red) (bottom). (D) SpiB mRNA expression in sorted IgG1 + and IgE + from iGB cells cultured with IL-4 (iGB-4) for 4 days and IL-21 (iGB-21) for the following 4 days. (E) FCM analysis of the expression of CD138 and CD38 in SpiB high (blue) and SpiB low (red) iGB-4 and iGB-21 cells. SpiB staining profile and gating of iGB-4 and iGB-21 cells are shown on the left and the expression of CD138 and CD38 on the gated fractions are on the right. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Intracellular SpiB staining was performed with FoxP3 Staining Buffer Set according to the manufacturer’s protocol (eBioscience) with anti-SpiB sheep polyclonal antibody (R&D systems, AF7204) followed by Alexa 647-labeled donkey anti-sheep IgG (Invitrogen, A-21448).

Techniques: In Vitro, Expressing, Comparison, Cell Culture, Immunofluorescence, Microscopy, Staining

Journal: Frontiers in Immunology

Article Title: SpiB regulates the expression of B-cell-related genes and increases the longevity of memory B cells

doi: 10.3389/fimmu.2023.1250719

Figure Lengend Snippet: SpiB suppresses PC differentiation and Blimp1 expression while upregulates Bach2 expression in vitro. (A) Experimental protocol for retroviral overexpression and knockdown of SpiB in iGB cells. Spleen B cells were cultured on 40LB feeder cells with IL-4 for 5 days (IGB-4), to which the overexpression or knockdown retroviral vectors were introduced on day 2, then followed by the culture with IL-21 for 3- 5 days (iGB-21). (B) FCM analysis of CD138 and IgE expression on SpiB-overexpressing (left) or SpiB-knockdown (right) IGB-4 and iGB-21 cells. (C) FCM analysis of Blimp1-GFP expression in SpiB-overexpressing (left) or SpiB-knockdown (right) IGB-4 and iGB-21 cells. IgE + , IgM + and IgE - IgM - (mostly IgG1 + ) populations were gated on iGB-4 and iGB-21 cells (top panels) and the expression of GFP in each population (bottom) were analyzed by FCM (bottom). (D, E) qRT-PCR analysis of mRNA expression of B cell-related genes in SpiB-overexpressng (D) and SpiB-knockdown (E) Blimp1-KO iGB-4 cells. *p < 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: Intracellular SpiB staining was performed with FoxP3 Staining Buffer Set according to the manufacturer’s protocol (eBioscience) with anti-SpiB sheep polyclonal antibody (R&D systems, AF7204) followed by Alexa 647-labeled donkey anti-sheep IgG (Invitrogen, A-21448).

Techniques: Expressing, In Vitro, Retroviral, Over Expression, Knockdown, Cell Culture, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: SpiB regulates the expression of B-cell-related genes and increases the longevity of memory B cells

doi: 10.3389/fimmu.2023.1250719

Figure Lengend Snippet: SpiB is critical for B mem cell generation from iGB cells in vivo. (A) Experimental protocol for the generation of iMB cells from gene-transduced iGB-4 cells. iGB-4 cells derived from Ly5.1 congenic C57BL/6 (B6) mice were transduced with retroviral vectors expressing SpiB or shSpiB, or empty vectors as a control (cont.). These iGB-4 cells on day 5 were adaptively transferred to irradiated Ly5.2 congenic B6 mice. Spleen cells were analyzed 4 weeks later (for SpiB overexpression) or 2 weeks later (for SpiB knockdown) by FCM. (B, C) Frequencies of CD19 + Ly5.1 + iMB cells in the spleens of the recipient mice. A representative FCM data (left) and the frequencies among lymphocytes (right) [ (B) : n = 6; (C) : n = 3]. *p < 0.05.

Article Snippet: Intracellular SpiB staining was performed with FoxP3 Staining Buffer Set according to the manufacturer’s protocol (eBioscience) with anti-SpiB sheep polyclonal antibody (R&D systems, AF7204) followed by Alexa 647-labeled donkey anti-sheep IgG (Invitrogen, A-21448).

Techniques: In Vivo, Derivative Assay, Transduction, Retroviral, Expressing, Control, Irradiation, Over Expression, Knockdown

Journal: Frontiers in Immunology

Article Title: SpiB regulates the expression of B-cell-related genes and increases the longevity of memory B cells

doi: 10.3389/fimmu.2023.1250719

Figure Lengend Snippet: Long-lived B mem cells and memory antibody response are defective in SpiB cKO mice. (A) Frequencies of CD19 + Ly5.1 + (total iMB) cells and CD19 + Ly5.1 + IgG1 + iMB cells in the spleens of the recipient mice transferred with iGB-4 cells derived from SpiB WT or SpiB cKO mouse B cells 4 weeks earlier, analyzed by FCM. A representative FCM data (left) and the frequencies among lymphocytes (right) (N =3). (B) A protocol of a mice immunization experiment. SpiB WT and SpiB cKO mice were intraperitoneally immunized with NP-CGG in alum and boosted with NP-GCC without alum to induce memory response at 66 days after the primary immunization. Spleen cells and blood were collected before immunization and at the indicated timepoints (spleen) and as indicated in (D) (blood). (C) Frequencies of NP + IgG1 + or NP + IgG1 - , total B, B mem and GC B cells in the spleens at the indicated time points after NP-CGG immunization in SpiB WT or SpiB cKO mice. Spleen cells were gated on B220 + Igκ low , NP + IgG1 + or NP + IgG1 - cells before gating on B mem (GL7 - CD38 + ) or GC B (GL7 + CD38 - ) cells. A representative FCM data (left) and the frequencies among lymphocytes of three to six mice (right) are shown. (D) Concentrations of serum antibodies of total anti-NP-IgG1 (left) and high-affinity anti-NP-IgG1 (right) from SpiB WT or SpiB cKO mice primarily immunized with NP-CGG in alum and secondarily with NP-CGG alone on day 66. (n = 4) *p < 0.05, **p < 0.01.

Article Snippet: Intracellular SpiB staining was performed with FoxP3 Staining Buffer Set according to the manufacturer’s protocol (eBioscience) with anti-SpiB sheep polyclonal antibody (R&D systems, AF7204) followed by Alexa 647-labeled donkey anti-sheep IgG (Invitrogen, A-21448).

Techniques: Derivative Assay

Journal: Frontiers in Immunology

Article Title: SpiB regulates the expression of B-cell-related genes and increases the longevity of memory B cells

doi: 10.3389/fimmu.2023.1250719

Figure Lengend Snippet: SpiB suppresses PC differentiation independently of Bach2. (A) Expression of intracellular SpiB and cell-surface CD138 in SpiB WT, Blimp1-KO, Bach2-KO and Blimp1/Bach2 DKO iGB-4 cells analyzed by FCM. (B) mRNA expression of SpiB , Bach2 , and Prdm1 in SpiB WT and SpiB cKO iGB-4 cells analyzed by qRT-PCR. (C) FCM analysis of CD138 expression on Bach2-KO iGB-4 (left) and iGB-21 (right) cells transduced with mock-, Bach2-, SpiB-expressing retroviral vectors. (D) qRT-PCR analysis for mRNA expression of SpiB , Bach2 , and Prdm1 in IgG1 + GC B and B mem cells from spleens of SpiB WT and SpiB cKO mice immunized with NP-CGG in alum 14 days earlier. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Intracellular SpiB staining was performed with FoxP3 Staining Buffer Set according to the manufacturer’s protocol (eBioscience) with anti-SpiB sheep polyclonal antibody (R&D systems, AF7204) followed by Alexa 647-labeled donkey anti-sheep IgG (Invitrogen, A-21448).

Techniques: Expressing, Quantitative RT-PCR, Transduction, Retroviral

Journal: Frontiers in Immunology

Article Title: SpiB regulates the expression of B-cell-related genes and increases the longevity of memory B cells

doi: 10.3389/fimmu.2023.1250719

Figure Lengend Snippet: SpiB-deficient GC B and B mem cells express lower levels of anti-apoptotic and autophagy genes and are more prone to die. (A, B) qRT-PCR analysis for mRNA expression of indicated anti-apoptotic genes (left) and autophagy genes (right) in IgG1 + GC B cells (A) and IgG1 + B mem cells (B) from spleens of SpiB WT and SpiB cKO mice immunized with NP-CGG in alum 14 days earlier. (C) FCM analysis for MitoSOX staining of the same cells as in (A, B) Cells were incubated for 0 or 9 hours in non-FCS medium before staining. Representative FCM data (left) and the frequencies of MitoSOX-positive cells (n = 3 or 4) (right). (D) FCM analysis for the expression of LC3B in the same cells as in (A, B) Representative FCM data (top) and mean fluorescence intensity (MFI) of the LC3B stained cells (bottom) are shown (n = 5). (E) Frequencies of dead cells in NP + IgG1 + CD38 + B mem cells from spleens of SpiB WT and SpiB cKO mice immunized with NP-CGG in alum 14 days earlier. Percentages of the dead cells detected microscopically following Trypan Blue staining after 9 hours’ culture in non-FCS medium as compared to those before the culture (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Intracellular SpiB staining was performed with FoxP3 Staining Buffer Set according to the manufacturer’s protocol (eBioscience) with anti-SpiB sheep polyclonal antibody (R&D systems, AF7204) followed by Alexa 647-labeled donkey anti-sheep IgG (Invitrogen, A-21448).

Techniques: Quantitative RT-PCR, Expressing, Staining, Incubation, Fluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: tsRNA-GlyGCC promotes colorectal cancer progression and 5-FU resistance by regulating SPIB

doi: 10.1186/s13046-024-03132-6

Figure Lengend Snippet: Primer sequence

Article Snippet: Relevant primary antibodies: GAPDH (CST #92,310), SPIB (15768-1-AP, Proteintech), BAX (380,709, zenbio), BCL2 (R23309, zenbio), EphB2 (83277-1-RR, Proteintech), L1CAM (R381761, zenbio), LGR5 (R380973, zenbio).

Techniques: Sequencing