Journal: Molecular & Cellular Proteomics

Article Title: Analysis of BCL6-interacting Proteins by Tandem Mass Spectrometry

doi: 10.1074/mcp.m500112-mcp200

Figure Lengend Snippet: FIG. 2. Experimental design. The BCL6 protein complex was iso- lated by antibody immunoprecipitation, and the proteins were sepa- rated by SDS-PAGE. The entire lane was submitted, in 12 separate gel slices, for trypsin digestion and analysis by ion trap tandem mass spectrometry.

Article Snippet: For immunoprecipitation, lysate containing 800 g of total protein was mixed with either 2 g of primary anti-BCL6 (C-19) polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or normal rabbit IgG in a total volume of 1 ml.

Techniques: Immunoprecipitation, SDS Page, Mass Spectrometry

Journal: Molecular & Cellular Proteomics

Article Title: Analysis of BCL6-interacting Proteins by Tandem Mass Spectrometry

doi: 10.1074/mcp.m500112-mcp200

Figure Lengend Snippet: FIG. 3. Immunoprecipitation of BCL6. Proteins immunoprecipi- tated from total cell lysates of SUDHL-4 and SUDHL-9 cell lines using a polyclonal anti-BCL6 antibody or rabbit IgG control were divided and resolved on parallel 8% SDS-polyacrylamide gels. A, silver stain- ing of one gel demonstrated a 95-kDa band corresponding to BCL6 that was present in the anti-BCL6 immunoprecipitate of SUDHL-4 lysates but not in the IgG immunoprecipitation control or the anti- BCL6 immunoprecipitate of SUDHL-9 lysates. B, after electrophore- sis, the proteins in the second gel were transferred to nitrocellulose membrane and immunoblotted using anti-BCL6 monoclonal anti- body. A band at 95 kDa corresponding to BCL6 was identified in the SUDHL-4 total cell lysate but not the SUDHL-9 or IgG control lanes.

Article Snippet: For immunoprecipitation, lysate containing 800 g of total protein was mixed with either 2 g of primary anti-BCL6 (C-19) polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or normal rabbit IgG in a total volume of 1 ml.

Techniques: Immunoprecipitation, Control, Silver Staining, Membrane

Journal: Molecular & Cellular Proteomics

Article Title: Analysis of BCL6-interacting Proteins by Tandem Mass Spectrometry

doi: 10.1074/mcp.m500112-mcp200

Figure Lengend Snippet: FIG. 1. BCL6-interacting proteins. The diagram shows the con- served motifs that mediate BCL6 protein interactions with the pro- teins listed under each domain. BCL6 directly interacts with several proteins via the POZ/BTB domain, the zinc finger domain, or both. p300 binds and acetylates BCL6, targeting it for degradation.

Article Snippet: For immunoprecipitation, lysate containing 800 g of total protein was mixed with either 2 g of primary anti-BCL6 (C-19) polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or normal rabbit IgG in a total volume of 1 ml.

Techniques:

Journal: Molecular & Cellular Proteomics

Article Title: Analysis of BCL6-interacting Proteins by Tandem Mass Spectrometry

doi: 10.1074/mcp.m500112-mcp200

Figure Lengend Snippet: FIG. 4. Protein identification by tan- dem mass spectrometry. MS/MS se- quencing of peptides identifying the pro- teins BCL6 (A), BCL11 (B), and PKC- (C). The MS full scan is shown for each sample followed by data-dependent MS/MS sequencing scans of a top pep- tide candidate.

Article Snippet: For immunoprecipitation, lysate containing 800 g of total protein was mixed with either 2 g of primary anti-BCL6 (C-19) polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or normal rabbit IgG in a total volume of 1 ml.

Techniques: Mass Spectrometry, Tandem Mass Spectroscopy, Sequencing

Journal: Molecular & Cellular Proteomics

Article Title: Analysis of BCL6-interacting Proteins by Tandem Mass Spectrometry

doi: 10.1074/mcp.m500112-mcp200

Figure Lengend Snippet: FIG. 5. Western blot and reciprocal immunoprecipitation valida- tion. A, proteins immunoprecipitated from SUDHL-4 lysates using anti-BCL6 were subjected to Western blotting using antibodies to the previously reported BCL6-interacting proteins HDAC4 and N-CoR2/ SMRT and the novel interacting proteins PKC- and WNK4. The bands were similar to those present in total cell lysates but were not seen in IgG immunoprecipitation controls. B, proteins immunoprecipi- tated from SUDHL-4 lysates using polyclonal antibodies to HDAC4 (previously reported BCL6-interacting protein) and PKC (novel inter- acting protein) were subjected to Western blotting using anti-BCL6. Bands correlating to BCL6 were identified by Western blotting in both pull-downs but not in the IgG control. IP, immunoprecipitation; WB, Western blot.

Article Snippet: For immunoprecipitation, lysate containing 800 g of total protein was mixed with either 2 g of primary anti-BCL6 (C-19) polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or normal rabbit IgG in a total volume of 1 ml.

Techniques: Western Blot, Immunoprecipitation, Control

Journal: Molecular & Cellular Proteomics

Article Title: Analysis of BCL6-interacting Proteins by Tandem Mass Spectrometry

doi: 10.1074/mcp.m500112-mcp200

Figure Lengend Snippet: FIG. 6. Cellular location and molecular function of BCL6-inter- acting proteins. The relative proportions of identified proteins are shown for the subcellular localization (A) and functional classification (B).

Article Snippet: For immunoprecipitation, lysate containing 800 g of total protein was mixed with either 2 g of primary anti-BCL6 (C-19) polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or normal rabbit IgG in a total volume of 1 ml.

Techniques: Functional Assay

Journal: Oncotarget

Article Title: BCL6 modulation of acute lymphoblastic leukemia response to chemotherapy

doi: 10.18632/oncotarget.8273

Figure Lengend Snippet: A. BCL6 protein in REH and Nalm-27 ALL cells when co-cultured with BMSC or HOB cells relative to media (M) controls as shown by western blot analysis. B. Flow cytometry analysis of REH and Nalm-27 ALL cell BCL6 protein levels when removed from the PD population compared to cells in media alone as shown by median florescence intensity (MFI). C. MFI of Patient 1 (P1) and Patient 2 (P2) when in physical contact with BMSC or HOB compared to those in media alone (ND = not detected). D. Confocal microscopy images of REH and Nalm-27 for BCL6 (yellow) and DAPI (Blue) in cells cultured in media alone compare to those recovered from the PD population of BMSC or HOB co-culture. E. P1 and P2 BCL6 confocal staining of media alone cells relative to those in contact with BMSC or HOB. Scale bar = 10μm.

Article Snippet: ALL cells were stained using Cell Signaling Technology's recommended protocol for intracellular BCL6 staining using primary antibodies rabbit anti-BCL6 (Cat # 14895) (1:300) or Rabbit (DA1E) mAb IgG XP isotype control (Cat # 3900).

Techniques: Cell Culture, Western Blot, Flow Cytometry, Confocal Microscopy, Co-Culture Assay, Staining

Journal: Oncotarget

Article Title: BCL6 modulation of acute lymphoblastic leukemia response to chemotherapy

doi: 10.18632/oncotarget.8273

Figure Lengend Snippet: A. - B. Cell density and viability of REH, Sup-B15, and Nalm-27 following exposure to the small molecule BCL6 inhibitor 79-6 (125μM) relative to DMSO controls as shown by trypan blue exclusion cell counts. C. Proliferation index of 79-6 treatment of REH, Sup-B15 and Nalm-27 ALL cells compared to DMSO controls using a CSFE cell retention dye flow cytometry analysis. D. Propidium iodide (PI) DNA staining for cell cycle assessment of REH, Sup-B15 and Nalm-27 treated with 79-6 compared to DMSO controls. E. Cell density of shRNA knockdown of BCL6 (KD1 and KD3) (left panel) and BCL6 overexpression (BCL6 OX) (right panel) of REH cells over time compared to vector controls as evaluated by trypan blue exclusion counts. F. Cell cycle analysis of BCL6 knockdown (left panel) and BCL6 overexpression (right panel) in REH cells using PI staining. (* = p < 0.05 for 79-6 treated cells or knockdown/overexpression cells compared to DMSO or vector controls, respectively).

Article Snippet: ALL cells were stained using Cell Signaling Technology's recommended protocol for intracellular BCL6 staining using primary antibodies rabbit anti-BCL6 (Cat # 14895) (1:300) or Rabbit (DA1E) mAb IgG XP isotype control (Cat # 3900).

Techniques: Flow Cytometry, Staining, shRNA, Knockdown, Over Expression, Plasmid Preparation, Cell Cycle Assay

Journal: Oncotarget

Article Title: BCL6 modulation of acute lymphoblastic leukemia response to chemotherapy

doi: 10.18632/oncotarget.8273

Figure Lengend Snippet: A. Western blot analysis of protein abundance of BCL6 and cyclin D3 in REH and Nalm-27 cells in media alone compared to PD cells recovered from BMSC or HOB co-culture. B. Comparison of REH BCL6 knockdown and overexpression to vector controls for BCL6 and cyclin D3 protein levels by western blot. C. Protein analysis by western blot of cyclin D3 in REH and Nalm-27 cells when exposed to 79-6.

Article Snippet: ALL cells were stained using Cell Signaling Technology's recommended protocol for intracellular BCL6 staining using primary antibodies rabbit anti-BCL6 (Cat # 14895) (1:300) or Rabbit (DA1E) mAb IgG XP isotype control (Cat # 3900).

Techniques: Western Blot, Quantitative Proteomics, Co-Culture Assay, Comparison, Knockdown, Over Expression, Plasmid Preparation

Journal: Oncotarget

Article Title: BCL6 modulation of acute lymphoblastic leukemia response to chemotherapy

doi: 10.18632/oncotarget.8273

Figure Lengend Snippet: A. Viability comparison of REH vector control, BCL6 overexpression, or BCL6 overexpression cells pre-treated with 79-6 (125μM) following exposure to three chemotherapy drugs (Ara-C [1 μM], MTX [50 μM], VCR [25 μM]). (* = p < 0.05 BCL6 OX to vector control and # = p < 0.05 BCL6 OX to BCL6 + 79-6). B. REH and Nalm-27 BCL6 protein dose response to MG132 and caffeine as shown by western blot. C. Western blot analysis to determine BCL6 protein abundance of REH and Nalm-27 cells when exposed to MG132 or caffeine when recovered from media alone, and the PD population of BMSC or HOB co-culture. D. REH and Nalm-27 cell viability following exposure to Ara-C alone or when pre-treated with MG132 or caffeine 6 hours prior to Ara-C exposure to compare cells in media alone to those recovered from the PD population of BMSC or HOB co-culture. (# = p < 0.05 PD ALL cells from BMSC/HOB relative to media and * = p < 0.05 Ara-C + MG132/Ara-C + caffeine relative to Ara-C only treatment).

Article Snippet: ALL cells were stained using Cell Signaling Technology's recommended protocol for intracellular BCL6 staining using primary antibodies rabbit anti-BCL6 (Cat # 14895) (1:300) or Rabbit (DA1E) mAb IgG XP isotype control (Cat # 3900).

Techniques: Comparison, Plasmid Preparation, Control, Over Expression, Western Blot, Quantitative Proteomics, Co-Culture Assay

Journal: Oncotarget

Article Title: BCL6 modulation of acute lymphoblastic leukemia response to chemotherapy

doi: 10.18632/oncotarget.8273

Figure Lengend Snippet: A. Schematic of NSG mouse experiment to determine GFP+ ALL burden in the femurs of NSG mice. B. Box plot representation of median percentage of GFP+ REH ALL cells relative to total mononuclear cells recovered from femurs of NOD-SCID Gamma (NSG) mice infected with REH vector control ( n = 5) or REH BCL6 overexpression ( n = 4) ALL cells following three consecutive days of Ara-C treatment. C. Schematic of NSG mouse experiment to determine event free survival of mice pre-treated with BCL6 modulating drugs MG-132 or caffeine. D. Event free survival of NSG mice following treatment with Ara-C ( n = 5), MG-132 + Ara-C ( n = 6), or caffeine + Ara-C ( n = 6) (* = p < 0.05 Ara-C relative to caffeine+ Ara-C).

Article Snippet: ALL cells were stained using Cell Signaling Technology's recommended protocol for intracellular BCL6 staining using primary antibodies rabbit anti-BCL6 (Cat # 14895) (1:300) or Rabbit (DA1E) mAb IgG XP isotype control (Cat # 3900).

Techniques: Infection, Plasmid Preparation, Control, Over Expression

Journal: Oncogene

Article Title: Prolactin suppresses a progestin-induced CK5-positive cell population in luminal breast cancer through inhibition of progestin-driven BCL6 expression

doi: 10.1038/onc.2013.172

Figure Lengend Snippet: (A) Immunoblots of BCL6, CK5, and GAPDH protein levels of T47D cells treated with the synthetic progestin R5020 (Pg) at the indicated doses for 24 h (top), or the indicated times with 20nM R5020 (Pg) (bottom). (B) Immunoblots of BCL6 protein in extracts of T47D cells treated with vehicle (Cntrl), Prolactin (PRL), β-Estradiol (E2), or R5020 (Pg) for the indicated times. (C) Immunoblots of BCL6, CK5, and GAPDH protein levels in extracts of T47D cells treated with hormones in the presence or absence of the progesterone receptor antagonist RU486 for 48 h. (D) Immunoblots of BCL6, phosphorylated-Stat5 (p-Stat5), and GAPDH protein levels in extracts of T47D, MCF-7, ZR75-1, BT-474, and HCC1937 cells treated with hormones for 72 h.

Article Snippet: The coverslips were then blocked with PBS+1% BSA before incubation with primary antibodies to BCL6, CK5, or Cleaved Caspase-3 (Cell Signaling Technology #9661), followed by secondary antibodies Alexa Fluor 555-conjugated goat anti-mouse antibody (Invitrogen) and Alexa Fluor 488-conjugated goat anti-rabbit antibody (Invitrogen).

Techniques: Western Blot

Journal: Oncogene

Article Title: Prolactin suppresses a progestin-induced CK5-positive cell population in luminal breast cancer through inhibition of progestin-driven BCL6 expression

doi: 10.1038/onc.2013.172

Figure Lengend Snippet: (A) Immunoblot of BCL6, CK5 and GAPDH in extracts of T47D and BT-474 cultures treated with non-target shRNA (NTC) or one of two independent BCL6 shRNAs (G2 and F11) for 48 h, followed by addition of vehicle (Cntrl), Prolactin (PRL), β-Estradiol (E2), or R5020 (Pg) for 48 h. (B) Quantification of CK5 immunocytochemistry (ICC) of T47D cultures exposed to either NTC shRNA or BCL6 shRNAs G2 or F11. Percent CK5 positive cells are indicated. In Pg treated cells, the percentage of CK5+ cells was 21.1 times higher (95% CI: 5.2, 31.1, P<0.001) under NTC condition as compared to G2 condition, and the percentage of CK5+ cells was 12.7 times higher (95% CI: 5.2, 31.1, P<0.001) under NTC condition as compared to F11 condition. (C) Representative images of ICC stained for CK5 (red) and DAPI (blue). (D) Representative images of ICC stained for BCL6 (red) and DAPI (blue).

Article Snippet: The coverslips were then blocked with PBS+1% BSA before incubation with primary antibodies to BCL6, CK5, or Cleaved Caspase-3 (Cell Signaling Technology #9661), followed by secondary antibodies Alexa Fluor 555-conjugated goat anti-mouse antibody (Invitrogen) and Alexa Fluor 488-conjugated goat anti-rabbit antibody (Invitrogen).

Techniques: Western Blot, shRNA, Immunocytochemistry, Staining

Journal: Oncogene

Article Title: Prolactin suppresses a progestin-induced CK5-positive cell population in luminal breast cancer through inhibition of progestin-driven BCL6 expression

doi: 10.1038/onc.2013.172

Figure Lengend Snippet: (A) Immunoblots of BCL6, CK5, phosphorylated-Stat5 (p-Stat5), Stat5a, and GAPDH protein levels in extracts of T47D cultures exposed to adenovirus carrying either Stat5a or LacZ control for 24 h and subsequently treated with vehicle (Cntrl), Prolactin (PRL), β-Estradiol (E2), or R5020 (Pg) for 48 h. (B) Immunoblots of BCL6, CK5, phosphorylated-Stat5 (p-Stat5), Stat5, Jak2, and GAPDH protein levels in extracts of T47D cultures treated with non-target shRNA (NTC) or Jak2 shRNA for 48h, followed by addition of vehicle (Cntrl), Prolactin (PRL), β-Estradiol (E2), or R5020 (Pg) for 48 h. (C) Immunoblots of BCL6, phosphorylated-ERK1/2 (p-ERK1/2), ERK1/2, and GAPDH protein levels in extracts of T47D cultures pre-treated with Pg for 24 h followed by incubation with or without prolactin (PRL) in the presence or absence of Mek inhibitor (U0126) for 3 and 6 h. (D) Immunoblots of BCL6, phosphorylated-Akt (p-Akt), Akt, and GAPDH protein levels in extracts of T47D cultures pre-treated with Pg for 24 h followed by incubation with or without prolactin (PRL) in the presence or absence of PI3K inhibitor (Ly294002) for 3 and 6 h.

Article Snippet: The coverslips were then blocked with PBS+1% BSA before incubation with primary antibodies to BCL6, CK5, or Cleaved Caspase-3 (Cell Signaling Technology #9661), followed by secondary antibodies Alexa Fluor 555-conjugated goat anti-mouse antibody (Invitrogen) and Alexa Fluor 488-conjugated goat anti-rabbit antibody (Invitrogen).

Techniques: Western Blot, Control, shRNA, Incubation

Journal: Oncogene

Article Title: Prolactin suppresses a progestin-induced CK5-positive cell population in luminal breast cancer through inhibition of progestin-driven BCL6 expression

doi: 10.1038/onc.2013.172

Figure Lengend Snippet: (A) Representative immunofluorescence images of breast cancer tissue sections of ER-positive, premenopausal patients stained for either BCL6 (red) or CK5 (red) and cytokeratin (green) and DAPI (blue). Case 1 represents a tumor with high CK5 and high BCL6, while Case 2 represents a patient with low CK5 and low BCL6. (B) Scatter plots and correlation of levels of nuclear BCL6 and cytoplasmic CK5 proteins in ER-positive tumors of patients younger than 50 years old at diagnosis (premenopausal; Spearman N=58, r=0.27, P=0.038). (C) Scatter plots and correlation of levels of nuclear BCL6 and cytoplasmic CK5 proteins in ER-positive tumors of patients older than 49 years at diagnosis (postmenopausal; Spearman N=164, r=−0.01, P=0.901). (D) Kaplan-Meier analysis of nuclear BCL6 in ER-positive tumors of patients younger than 50 years of age (Logrank P=0.044). (E) Kaplan-Meir analysis of cytoplasmic CK5 in ER-positive tumors of patients younger than 50 years of age (Logrank P=0.033).

Article Snippet: The coverslips were then blocked with PBS+1% BSA before incubation with primary antibodies to BCL6, CK5, or Cleaved Caspase-3 (Cell Signaling Technology #9661), followed by secondary antibodies Alexa Fluor 555-conjugated goat anti-mouse antibody (Invitrogen) and Alexa Fluor 488-conjugated goat anti-rabbit antibody (Invitrogen).

Techniques: Immunofluorescence, Staining, Biomarker Discovery

Journal: PLOS Pathogens

Article Title: RACK1 enhances STAT3 stability and promotes T follicular helper cell development and function during blood-stage Plasmodium infection in mice

doi: 10.1371/journal.ppat.1012352

Figure Lengend Snippet: (A) Representative counter plots and bar graphs showing the proportions and numbers of Tfh (CD4 + CXCR5 + PD-1 + ) cells in the spleen of WT and RACK1 KO mice at day 0, day 7, and day 16 p.i. (B) Representative counter plots and bar graphs showing the proportions and numbers of GC Tfh (CD4 + CXCR5 + Bcl-6 hi ) cells in the spleen of WT and KO mice at day 7 and day 16 p.i. (C) Quantitative RT-PCR analysis of Tfh-lineage signature genes Bcl-6 , Cxcr5 , Pdcd1 , Il-21 , Icos and special regulatory genes Tcf7 , Prdm1 , sh2d1a , S1pr1 in splenic CD4 + T cells from WT and KO mice at day 6 p.i., bar graph showing gene expression level normalized to β-actin , relative to that of WT mice. (D) IB analysis of Bcl-6 expression in splenic CD4 + T cells from WT and KO mice at day 6 p.i. (n = 4 mice/group). Numbers indicate densitometry of the bands normalized to β-actin , relative to that of WT mice. (E) Gating strategy of CXCR3, CCR6 expression on CXCR5 + CD4 + T cells, bar graph showing the proportions of Th1-like (CXCR3 + CCR6 - ), Th2-like (CXCR3 - CCR6 - ) and Th17-like (CXCR3 - CCR6 + ) Tfh subsets in the spleen of WT and KO mice at day 7 p.i. Data are from three or four experiments with 6–12 mice/group (A, B) or representative of two replicate experiments with 3–6 mice/group (C-E). Data are shown as mean±SD. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 and ns, not significant by Mann-Whitney test (A, B) or Student’s t test (C, E).

Article Snippet: Intracellular transcription factor staining was performed using Foxp3/transcription factor staining buffer set (eBioscience), APC-conjugated anti-Foxp3 (eBioscience), primary antibodies against Bcl-6 (BD Pharmingen) and STAT3 (Proteintech), and appropriate secondary antibodies conjugated with FITC (ABclonal).

Techniques: Quantitative RT-PCR, Expressing, MANN-WHITNEY

Journal: PLOS Pathogens

Article Title: RACK1 enhances STAT3 stability and promotes T follicular helper cell development and function during blood-stage Plasmodium infection in mice

doi: 10.1371/journal.ppat.1012352

Figure Lengend Snippet: (A) Naïve CD4 + T cells from WT and RACK1 KO mice were cultured under the Tfh-like skewing condition for 3 days, with or without adding 1μM STAT3 activity inhibitor, niclosamide (Ni). The pSTAT3 (Tyr 705 ) levels in naïve and Tfh-like differentiated cells were examined by IB analysis, numbers indicate densitometry of the bands relative to that of naïve WT cells. (B) Quantitative RT-PCR analysis of Bcl-6 expression in naïve and Tfh-like differentiated CD4 + T cells as described in (A) (n = 3 mice/group). (C-E) Naïve CD4 + T cells sorted from WT and RACK1 KO mice were pre-activated and spin infected with the retrovirus expressing RACK1 (OE-Rack1) or GFP alone (EV), the transduced (GFP + ) cells were then sorted and polarized under the Tfh-like condition for 3 days. (C) Validation of RACK1 expression in retrovirally transduced GFP + CD4 + T cells, numbers indicate densitometry of the bands relative to that of WT cells transduced with EV. (D) Phosflow analysis of pSTAT3 (Tyr 705 ) MFI in Tfh-like differentiated cells (36 h) transduced with the OE-Rack1 or EV vectors, the dash lines depict isotype IgG staining controls. (E) Quantitative RT-PCR for Bcl-6 expression in Tfh-like differentiated cells (3 days) transduced with the OE-Rack1 or EV vectors, data shown are Bcl-6 mRNA levels normalized to β-actin , relative to that of WT cells transduced with EV. Data are representative of two independent experiments and are shown as mean±SD. ** P <0.01, *** P <0.001, **** P <0.0001 and ns, not significant by two-way ANOVA with Bonferroni’s test (B) or one-way ANOVA with Tukey’s test (E).

Article Snippet: Intracellular transcription factor staining was performed using Foxp3/transcription factor staining buffer set (eBioscience), APC-conjugated anti-Foxp3 (eBioscience), primary antibodies against Bcl-6 (BD Pharmingen) and STAT3 (Proteintech), and appropriate secondary antibodies conjugated with FITC (ABclonal).

Techniques: Cell Culture, Activity Assay, Quantitative RT-PCR, Expressing, Infection, Transduction, Staining

Journal: PLOS Pathogens

Article Title: RACK1 enhances STAT3 stability and promotes T follicular helper cell development and function during blood-stage Plasmodium infection in mice

doi: 10.1371/journal.ppat.1012352

Figure Lengend Snippet: (A-C) Naïve CD4 + T cells from WT and RACK1 KO mice were transduced with the STAT3 overexpression retroviral vector (OE-Stat3) or GFP-expressing alone empty vector (EV), the transduced (GFP + ) cells were then polarized under the Tfh-like condition for 3 days. (A) IB analysis of STAT3 protein levels in the retrovirally transduced GFP + CD4 + T cells. (B) Rrepresentative histograms showing pSTAT3 (Tyr 705 ) in Tfh-like differentiated cells (48 h) transduced with the OE-Stat3 or EV vectors, the dash lines depict isotype IgG staining controls. Numbers indicate MFI of pSTAT3 in gated populations. (C) Quantitative RT-PCR for Bcl-6 expression in Tfh-like differentiated cells (n = 3/group). (D) Quantitative RT-PCR for Stat3 mRNA in splenic CD4 + T cells from WT and RACK1 KO mice at day 5 p.i. (n = 4-5/group). (E) Left, splenic CD4 + T cells from day 5-infected WT and RACK1 KO mice were treated with 10 μg/ml cycloheximide (CHX) for 0, 2, 4, 6 h, then subjected to IB analysis of STAT3, numbers indicate densitometry of the bands normalized to β-actin, relative to untreated cells (0 h). Right, summary graph showing quantification of relative STAT3 protein levels conducted by three independent experiments. (F) IB analysis of STAT3 in CD4 + T cells from day 5-infected WT and RACK1 KO mice treated with 20 μM MG132, 100 μM chloroquine (CQ), or vehicle (DMSO) for 8 h, respectively. Numbers showing band density normalized to β-actin, relative to WT cells treated with DMSO. (G) Splenic CD4 + T cells from day 5-infected WT and RACK1 KO mice were treated with 20 μM MG132 or DMSO for 8 h, endogenous ubiquitination of STAT3 was analyzed by IP with anti-STAT3 antibody, followed by IB with anti-ubiquitin (Ub) antibody. Numbers indicate densitometry of ubiquitinated-STAT3 normalized to the total amount of STAT3 in the immunoprecipitates. Data are representative of two or three independent experiments. * P <0.05, ** P <0.01, *** P <0.001 and ns, not significant by one-way ANOVA with Tukey’s test (C), Student’s t test (D), two-way ANOVA with Bonferroni’s test (E).

Article Snippet: Intracellular transcription factor staining was performed using Foxp3/transcription factor staining buffer set (eBioscience), APC-conjugated anti-Foxp3 (eBioscience), primary antibodies against Bcl-6 (BD Pharmingen) and STAT3 (Proteintech), and appropriate secondary antibodies conjugated with FITC (ABclonal).

Techniques: Transduction, Over Expression, Retroviral, Plasmid Preparation, Expressing, Staining, Quantitative RT-PCR, Infection

Journal: PLOS Pathogens

Article Title: RACK1 enhances STAT3 stability and promotes T follicular helper cell development and function during blood-stage Plasmodium infection in mice

doi: 10.1371/journal.ppat.1012352

Figure Lengend Snippet: (A) The interactions between STAT3 and Wwp2 (or Itch) in 293T cells transfected with plasmids expressing Myc-tagged Stat3 and Flag-tagged Wwp2 (or Itch ) were detected by IP with an anti-Flag antibody, followed by IB with an anti-Myc antibody. Lysates without IP were prepared for IB of plasmid expression. (B) Ubiquitination of STAT3 in 293T cells transfected with Myc-tagged Stat3 and HA-tagged Ubiquitin (Ub), with or without Flag-tagged Wwp2 (or Itch ) co-transfection was analyzed by IP with an anti-Myc antibody, followed by IB with an Ubiquitin antibody. MG132 was added for the last 8 h before cell collection. Numbers indicate densitometry of ubiquitinated-Myc-STAT3 normalized to the total amount of Myc-STAT3 in the immunoprecipitates. (C-D) Endogenous interaction between STAT3 and Wwp2 (or Itch) in CD4 + T cells from WT and RACK1 KO mice at day 5 p.i. was determined by IP with anti-STAT3 antibody, followed by IB with anti-Wwp2 (or anti-Itch) antibodies. Lysates without IP were prepared for IB analysis of STAT3 and Wwp2 (or Itch) expression. (E) CD4 + T cells from WT and RACK1 KO mice were transduced with the retroviral vectors expressing shRNA targeted Wwp2 (sh- Wwp2 ), Itch (sh- Itch ), or GFP alone (EV), the GFP + cells were then polarized under the Tfh-like condition. Left, quantitative RT-PCR for Wwp2 (or Itch ) mRNA in GFP + CD4 + T cells. Right, representative histograms showing the MFI of STAT3 in Tfh-like differentiated cells (24 h) retrovirally transduced with sh- Wwp2 , sh- Itch , or EV, the dash lines depict isotype IgG staining controls. (F) Schematic diagram showing the mechanism of RACK1-regulated Tfh differentiation and function. RACK1 may work to prevent the attachment of certain E3 ligases to STAT3 and subsequent degradation of STAT3 by the ubiquitin-proteasome system. This leads to enhanced stability and phosphorylation activity of STAT3 in CD4 + T cells, which ultimately promotes Bcl-6 induction, Tfh development, and generation of parasite-specific antibodies during P . yoelii infection. Data in (A-E) are representative of two independent experiments with similar results.

Article Snippet: Intracellular transcription factor staining was performed using Foxp3/transcription factor staining buffer set (eBioscience), APC-conjugated anti-Foxp3 (eBioscience), primary antibodies against Bcl-6 (BD Pharmingen) and STAT3 (Proteintech), and appropriate secondary antibodies conjugated with FITC (ABclonal).

Techniques: Transfection, Expressing, Plasmid Preparation, Cotransfection, Transduction, Retroviral, shRNA, Quantitative RT-PCR, Staining, Activity Assay, Infection