electrophoretic mobility shift assay recombinant human tcl1a protein Search Results


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oligonucleotides  (Jackson Laboratory)
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Figure 4. BAFFR regulates the expression of PTEN and FOXO1 (A) Western blot analysis of PTEN and FOXO1 in naive and MBCs treated for 24–72 h ± BAFF (20 ng/mL). N-fold changes of PTEN and FOXO1 expression were calculated as signals normalized to actin and further normalized to untreated samples from day 0 per subset. The experiment was performed once in this format. (B) Flow cytometric analysis of PTEN expression in naive and MBCs treated for 24–72 h ± BAFF. N-fold change of PTEN MFI was calculated as [(PTEN MFI + BAFF)/(PTEN MFI –BAFF)]. (C) Western blot analysis of phosphorylated FOXO1 (S256) in naive B cells treated with BAFF or F(ab0)2 anti-IgM (2 mg/mL) for 0.5–8 h. N-fold change in pFOXO1 levels was calculated as described in (A). (D) Western blot analysis of PTEN in naive B cells treated with inhibitors against SYK (2.5 mM R406), PI3Kd (0.625 mM nemiralisib), PDK1 (5 mM BX-795), <t>NIK</t> (10 mM <t>SMI1),</t> or DMSO (control) ± BAFF for 2 days. (E) Western blot analysis of FOXO1 in naive B cells treated with iSYK, iNIK, or DMSO ± BAFF for 2 days. (D and E) Blots are representative of R2 independent experiments. N-fold changes were calculated as signals normalized to actin and further normalized to the control untreated sample. (F) Flow cytometric analysis of CD62L and CXCR4 in naive and MBCs treated overnight with BAFF in the presence of FOXO1 inhibitor (2.5 mM AS1842856) or DMSO (control). Plots show the mean of CD62L and CXCR4 MFI from 2 independent experiments (2–3 replicates/experiment, 2 HDs). *p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant (2-way ANOVA with Tukey multiple comparisons test).
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Figure 4. BAFFR regulates the expression of PTEN and FOXO1 (A) Western blot analysis of PTEN and FOXO1 in naive and MBCs treated for 24–72 h ± BAFF (20 ng/mL). N-fold changes of PTEN and FOXO1 expression were calculated as signals normalized to actin and further normalized to untreated samples from day 0 per subset. The experiment was performed once in this format. (B) Flow cytometric analysis of PTEN expression in naive and MBCs treated for 24–72 h ± BAFF. N-fold change of PTEN MFI was calculated as [(PTEN MFI + BAFF)/(PTEN MFI –BAFF)]. (C) Western blot analysis of phosphorylated FOXO1 (S256) in naive B cells treated with BAFF or F(ab0)2 anti-IgM (2 mg/mL) for 0.5–8 h. N-fold change in pFOXO1 levels was calculated as described in (A). (D) Western blot analysis of PTEN in naive B cells treated with inhibitors against SYK (2.5 mM R406), PI3Kd (0.625 mM nemiralisib), PDK1 (5 mM BX-795), <t>NIK</t> (10 mM <t>SMI1),</t> or DMSO (control) ± BAFF for 2 days. (E) Western blot analysis of FOXO1 in naive B cells treated with iSYK, iNIK, or DMSO ± BAFF for 2 days. (D and E) Blots are representative of R2 independent experiments. N-fold changes were calculated as signals normalized to actin and further normalized to the control untreated sample. (F) Flow cytometric analysis of CD62L and CXCR4 in naive and MBCs treated overnight with BAFF in the presence of FOXO1 inhibitor (2.5 mM AS1842856) or DMSO (control). Plots show the mean of CD62L and CXCR4 MFI from 2 independent experiments (2–3 replicates/experiment, 2 HDs). *p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant (2-way ANOVA with Tukey multiple comparisons test).
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Figure 4. BAFFR regulates the expression of PTEN and FOXO1 (A) Western blot analysis of PTEN and FOXO1 in naive and MBCs treated for 24–72 h ± BAFF (20 ng/mL). N-fold changes of PTEN and FOXO1 expression were calculated as signals normalized to actin and further normalized to untreated samples from day 0 per subset. The experiment was performed once in this format. (B) Flow cytometric analysis of PTEN expression in naive and MBCs treated for 24–72 h ± BAFF. N-fold change of PTEN MFI was calculated as [(PTEN MFI + BAFF)/(PTEN MFI –BAFF)]. (C) Western blot analysis of phosphorylated FOXO1 (S256) in naive B cells treated with BAFF or F(ab0)2 anti-IgM (2 mg/mL) for 0.5–8 h. N-fold change in pFOXO1 levels was calculated as described in (A). (D) Western blot analysis of PTEN in naive B cells treated with inhibitors against SYK (2.5 mM R406), PI3Kd (0.625 mM nemiralisib), PDK1 (5 mM BX-795), <t>NIK</t> (10 mM <t>SMI1),</t> or DMSO (control) ± BAFF for 2 days. (E) Western blot analysis of FOXO1 in naive B cells treated with iSYK, iNIK, or DMSO ± BAFF for 2 days. (D and E) Blots are representative of R2 independent experiments. N-fold changes were calculated as signals normalized to actin and further normalized to the control untreated sample. (F) Flow cytometric analysis of CD62L and CXCR4 in naive and MBCs treated overnight with BAFF in the presence of FOXO1 inhibitor (2.5 mM AS1842856) or DMSO (control). Plots show the mean of CD62L and CXCR4 MFI from 2 independent experiments (2–3 replicates/experiment, 2 HDs). *p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant (2-way ANOVA with Tukey multiple comparisons test).
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cell signaling technology 8803  (Cell Signaling Technology Inc)
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Figure 4. BAFFR regulates the expression of PTEN and FOXO1 (A) Western blot analysis of PTEN and FOXO1 in naive and MBCs treated for 24–72 h ± BAFF (20 ng/mL). N-fold changes of PTEN and FOXO1 expression were calculated as signals normalized to actin and further normalized to untreated samples from day 0 per subset. The experiment was performed once in this format. (B) Flow cytometric analysis of PTEN expression in naive and MBCs treated for 24–72 h ± BAFF. N-fold change of PTEN MFI was calculated as [(PTEN MFI + BAFF)/(PTEN MFI –BAFF)]. (C) Western blot analysis of phosphorylated FOXO1 (S256) in naive B cells treated with BAFF or F(ab0)2 anti-IgM (2 mg/mL) for 0.5–8 h. N-fold change in pFOXO1 levels was calculated as described in (A). (D) Western blot analysis of PTEN in naive B cells treated with inhibitors against SYK (2.5 mM R406), PI3Kd (0.625 mM nemiralisib), PDK1 (5 mM BX-795), <t>NIK</t> (10 mM <t>SMI1),</t> or DMSO (control) ± BAFF for 2 days. (E) Western blot analysis of FOXO1 in naive B cells treated with iSYK, iNIK, or DMSO ± BAFF for 2 days. (D and E) Blots are representative of R2 independent experiments. N-fold changes were calculated as signals normalized to actin and further normalized to the control untreated sample. (F) Flow cytometric analysis of CD62L and CXCR4 in naive and MBCs treated overnight with BAFF in the presence of FOXO1 inhibitor (2.5 mM AS1842856) or DMSO (control). Plots show the mean of CD62L and CXCR4 MFI from 2 independent experiments (2–3 replicates/experiment, 2 HDs). *p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant (2-way ANOVA with Tukey multiple comparisons test).
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cas 702675 74 9 foxo1 inhibitor as1842856 selleckchem  (Selleck Chemicals)
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Figure 4. BAFFR regulates the expression of PTEN and <t>FOXO1</t> (A) Western blot analysis of PTEN and FOXO1 in naive and MBCs treated for 24–72 h ± BAFF (20 ng/mL). N-fold changes of PTEN and FOXO1 expression were calculated as signals normalized to actin and further normalized to untreated samples from day 0 per subset. The experiment was performed once in this format. (B) Flow cytometric analysis of PTEN expression in naive and MBCs treated for 24–72 h ± BAFF. N-fold change of PTEN MFI was calculated as [(PTEN MFI + BAFF)/(PTEN MFI –BAFF)]. (C) Western blot analysis of phosphorylated FOXO1 (S256) in naive B cells treated with BAFF or F(ab0)2 anti-IgM (2 mg/mL) for 0.5–8 h. N-fold change in pFOXO1 levels was calculated as described in (A). (D) Western blot analysis of PTEN in naive B cells treated with inhibitors against SYK (2.5 mM R406), PI3Kd (0.625 mM nemiralisib), PDK1 (5 mM BX-795), NIK (10 mM SMI1), or DMSO (control) ± BAFF for 2 days. (E) Western blot analysis of FOXO1 in naive B cells treated with iSYK, iNIK, or DMSO ± BAFF for 2 days. (D and E) Blots are representative of R2 independent experiments. N-fold changes were calculated as signals normalized to actin and further normalized to the control untreated sample. (F) Flow cytometric analysis of CD62L and CXCR4 in naive and MBCs treated overnight with BAFF in the presence of FOXO1 inhibitor (2.5 mM <t>AS1842856)</t> or DMSO (control). Plots show the mean of CD62L and CXCR4 MFI from 2 independent experiments (2–3 replicates/experiment, 2 HDs). *p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant (2-way ANOVA with Tukey multiple comparisons test).
Cas 702675 74 9 Foxo1 Inhibitor As1842856 Selleckchem, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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recombinant dna il4i1 cdna  (MyBiosource Biotechnology)
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MyBiosource Biotechnology recombinant dna il4i1 cdna
Figure 4. BAFFR regulates the expression of PTEN and <t>FOXO1</t> (A) Western blot analysis of PTEN and FOXO1 in naive and MBCs treated for 24–72 h ± BAFF (20 ng/mL). N-fold changes of PTEN and FOXO1 expression were calculated as signals normalized to actin and further normalized to untreated samples from day 0 per subset. The experiment was performed once in this format. (B) Flow cytometric analysis of PTEN expression in naive and MBCs treated for 24–72 h ± BAFF. N-fold change of PTEN MFI was calculated as [(PTEN MFI + BAFF)/(PTEN MFI –BAFF)]. (C) Western blot analysis of phosphorylated FOXO1 (S256) in naive B cells treated with BAFF or F(ab0)2 anti-IgM (2 mg/mL) for 0.5–8 h. N-fold change in pFOXO1 levels was calculated as described in (A). (D) Western blot analysis of PTEN in naive B cells treated with inhibitors against SYK (2.5 mM R406), PI3Kd (0.625 mM nemiralisib), PDK1 (5 mM BX-795), NIK (10 mM SMI1), or DMSO (control) ± BAFF for 2 days. (E) Western blot analysis of FOXO1 in naive B cells treated with iSYK, iNIK, or DMSO ± BAFF for 2 days. (D and E) Blots are representative of R2 independent experiments. N-fold changes were calculated as signals normalized to actin and further normalized to the control untreated sample. (F) Flow cytometric analysis of CD62L and CXCR4 in naive and MBCs treated overnight with BAFF in the presence of FOXO1 inhibitor (2.5 mM <t>AS1842856)</t> or DMSO (control). Plots show the mean of CD62L and CXCR4 MFI from 2 independent experiments (2–3 replicates/experiment, 2 HDs). *p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant (2-way ANOVA with Tukey multiple comparisons test).
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s1274  (Selleck Chemicals)
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Figure 4. BAFFR regulates the expression of PTEN and <t>FOXO1</t> (A) Western blot analysis of PTEN and FOXO1 in naive and MBCs treated for 24–72 h ± BAFF (20 ng/mL). N-fold changes of PTEN and FOXO1 expression were calculated as signals normalized to actin and further normalized to untreated samples from day 0 per subset. The experiment was performed once in this format. (B) Flow cytometric analysis of PTEN expression in naive and MBCs treated for 24–72 h ± BAFF. N-fold change of PTEN MFI was calculated as [(PTEN MFI + BAFF)/(PTEN MFI –BAFF)]. (C) Western blot analysis of phosphorylated FOXO1 (S256) in naive B cells treated with BAFF or F(ab0)2 anti-IgM (2 mg/mL) for 0.5–8 h. N-fold change in pFOXO1 levels was calculated as described in (A). (D) Western blot analysis of PTEN in naive B cells treated with inhibitors against SYK (2.5 mM R406), PI3Kd (0.625 mM nemiralisib), PDK1 (5 mM BX-795), NIK (10 mM SMI1), or DMSO (control) ± BAFF for 2 days. (E) Western blot analysis of FOXO1 in naive B cells treated with iSYK, iNIK, or DMSO ± BAFF for 2 days. (D and E) Blots are representative of R2 independent experiments. N-fold changes were calculated as signals normalized to actin and further normalized to the control untreated sample. (F) Flow cytometric analysis of CD62L and CXCR4 in naive and MBCs treated overnight with BAFF in the presence of FOXO1 inhibitor (2.5 mM <t>AS1842856)</t> or DMSO (control). Plots show the mean of CD62L and CXCR4 MFI from 2 independent experiments (2–3 replicates/experiment, 2 HDs). *p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant (2-way ANOVA with Tukey multiple comparisons test).
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s2194  (Selleck Chemicals)
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Figure 4. BAFFR regulates the expression of PTEN and <t>FOXO1</t> (A) Western blot analysis of PTEN and FOXO1 in naive and MBCs treated for 24–72 h ± BAFF (20 ng/mL). N-fold changes of PTEN and FOXO1 expression were calculated as signals normalized to actin and further normalized to untreated samples from day 0 per subset. The experiment was performed once in this format. (B) Flow cytometric analysis of PTEN expression in naive and MBCs treated for 24–72 h ± BAFF. N-fold change of PTEN MFI was calculated as [(PTEN MFI + BAFF)/(PTEN MFI –BAFF)]. (C) Western blot analysis of phosphorylated FOXO1 (S256) in naive B cells treated with BAFF or F(ab0)2 anti-IgM (2 mg/mL) for 0.5–8 h. N-fold change in pFOXO1 levels was calculated as described in (A). (D) Western blot analysis of PTEN in naive B cells treated with inhibitors against SYK (2.5 mM R406), PI3Kd (0.625 mM nemiralisib), PDK1 (5 mM BX-795), NIK (10 mM SMI1), or DMSO (control) ± BAFF for 2 days. (E) Western blot analysis of FOXO1 in naive B cells treated with iSYK, iNIK, or DMSO ± BAFF for 2 days. (D and E) Blots are representative of R2 independent experiments. N-fold changes were calculated as signals normalized to actin and further normalized to the control untreated sample. (F) Flow cytometric analysis of CD62L and CXCR4 in naive and MBCs treated overnight with BAFF in the presence of FOXO1 inhibitor (2.5 mM <t>AS1842856)</t> or DMSO (control). Plots show the mean of CD62L and CXCR4 MFI from 2 independent experiments (2–3 replicates/experiment, 2 HDs). *p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant (2-way ANOVA with Tukey multiple comparisons test).
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s1369  (Selleck Chemicals)
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Figure 4. BAFFR regulates the expression of PTEN and <t>FOXO1</t> (A) Western blot analysis of PTEN and FOXO1 in naive and MBCs treated for 24–72 h ± BAFF (20 ng/mL). N-fold changes of PTEN and FOXO1 expression were calculated as signals normalized to actin and further normalized to untreated samples from day 0 per subset. The experiment was performed once in this format. (B) Flow cytometric analysis of PTEN expression in naive and MBCs treated for 24–72 h ± BAFF. N-fold change of PTEN MFI was calculated as [(PTEN MFI + BAFF)/(PTEN MFI –BAFF)]. (C) Western blot analysis of phosphorylated FOXO1 (S256) in naive B cells treated with BAFF or F(ab0)2 anti-IgM (2 mg/mL) for 0.5–8 h. N-fold change in pFOXO1 levels was calculated as described in (A). (D) Western blot analysis of PTEN in naive B cells treated with inhibitors against SYK (2.5 mM R406), PI3Kd (0.625 mM nemiralisib), PDK1 (5 mM BX-795), NIK (10 mM SMI1), or DMSO (control) ± BAFF for 2 days. (E) Western blot analysis of FOXO1 in naive B cells treated with iSYK, iNIK, or DMSO ± BAFF for 2 days. (D and E) Blots are representative of R2 independent experiments. N-fold changes were calculated as signals normalized to actin and further normalized to the control untreated sample. (F) Flow cytometric analysis of CD62L and CXCR4 in naive and MBCs treated overnight with BAFF in the presence of FOXO1 inhibitor (2.5 mM <t>AS1842856)</t> or DMSO (control). Plots show the mean of CD62L and CXCR4 MFI from 2 independent experiments (2–3 replicates/experiment, 2 HDs). *p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant (2-way ANOVA with Tukey multiple comparisons test).
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Image Search Results


Figure 4. BAFFR regulates the expression of PTEN and FOXO1 (A) Western blot analysis of PTEN and FOXO1 in naive and MBCs treated for 24–72 h ± BAFF (20 ng/mL). N-fold changes of PTEN and FOXO1 expression were calculated as signals normalized to actin and further normalized to untreated samples from day 0 per subset. The experiment was performed once in this format. (B) Flow cytometric analysis of PTEN expression in naive and MBCs treated for 24–72 h ± BAFF. N-fold change of PTEN MFI was calculated as [(PTEN MFI + BAFF)/(PTEN MFI –BAFF)]. (C) Western blot analysis of phosphorylated FOXO1 (S256) in naive B cells treated with BAFF or F(ab0)2 anti-IgM (2 mg/mL) for 0.5–8 h. N-fold change in pFOXO1 levels was calculated as described in (A). (D) Western blot analysis of PTEN in naive B cells treated with inhibitors against SYK (2.5 mM R406), PI3Kd (0.625 mM nemiralisib), PDK1 (5 mM BX-795), NIK (10 mM SMI1), or DMSO (control) ± BAFF for 2 days. (E) Western blot analysis of FOXO1 in naive B cells treated with iSYK, iNIK, or DMSO ± BAFF for 2 days. (D and E) Blots are representative of R2 independent experiments. N-fold changes were calculated as signals normalized to actin and further normalized to the control untreated sample. (F) Flow cytometric analysis of CD62L and CXCR4 in naive and MBCs treated overnight with BAFF in the presence of FOXO1 inhibitor (2.5 mM AS1842856) or DMSO (control). Plots show the mean of CD62L and CXCR4 MFI from 2 independent experiments (2–3 replicates/experiment, 2 HDs). *p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant (2-way ANOVA with Tukey multiple comparisons test).

Journal: Cell reports

Article Title: BAFFR activates PI3K/AKT signaling in human naive but not in switched memory B cells through direct interactions with B cell antigen receptors.

doi: 10.1016/j.celrep.2022.111019

Figure Lengend Snippet: Figure 4. BAFFR regulates the expression of PTEN and FOXO1 (A) Western blot analysis of PTEN and FOXO1 in naive and MBCs treated for 24–72 h ± BAFF (20 ng/mL). N-fold changes of PTEN and FOXO1 expression were calculated as signals normalized to actin and further normalized to untreated samples from day 0 per subset. The experiment was performed once in this format. (B) Flow cytometric analysis of PTEN expression in naive and MBCs treated for 24–72 h ± BAFF. N-fold change of PTEN MFI was calculated as [(PTEN MFI + BAFF)/(PTEN MFI –BAFF)]. (C) Western blot analysis of phosphorylated FOXO1 (S256) in naive B cells treated with BAFF or F(ab0)2 anti-IgM (2 mg/mL) for 0.5–8 h. N-fold change in pFOXO1 levels was calculated as described in (A). (D) Western blot analysis of PTEN in naive B cells treated with inhibitors against SYK (2.5 mM R406), PI3Kd (0.625 mM nemiralisib), PDK1 (5 mM BX-795), NIK (10 mM SMI1), or DMSO (control) ± BAFF for 2 days. (E) Western blot analysis of FOXO1 in naive B cells treated with iSYK, iNIK, or DMSO ± BAFF for 2 days. (D and E) Blots are representative of R2 independent experiments. N-fold changes were calculated as signals normalized to actin and further normalized to the control untreated sample. (F) Flow cytometric analysis of CD62L and CXCR4 in naive and MBCs treated overnight with BAFF in the presence of FOXO1 inhibitor (2.5 mM AS1842856) or DMSO (control). Plots show the mean of CD62L and CXCR4 MFI from 2 independent experiments (2–3 replicates/experiment, 2 HDs). *p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant (2-way ANOVA with Tukey multiple comparisons test).

Article Snippet: Chemicals, peptides, and recombinant proteins BAFF Smulski et al., 2017 N/A CD40L Smulski et al., 2017 N/A SARS-Cov2 spike protein Thermofisher Cat# RP-87680 SYK inhibitor (R406) Selleckchem Cat# S2194; CAS 841290-81-1 Dual BCR-ABL/LYN inhibitor (Bafetinib/INNO-406) Selleckchem Cat# S1369; CAS 859212-16-1 PI3Kd inhibitor (nemiralisib/GSK2269557) Selleckchem Cat# S7937; CAS 1254036-71-9 PDK1 inhibitor (BX-795) Selleckchem Cat# S1274; CAS 702675-74-9 FOXO1 inhibitor (AS1842856) Selleckchem Cat# S8222; CAS 836620-48-5 NIK inhibitor (SMI1) Hycultec Cat# HY112433; CAS 1660114-31-7 human monoclonal IgG1l anti-BAFF antibody belimumab (Benlysta) GlaxoSmithKline Pharmaceuticals CAS 356547-88-1 hTACI (aa 31–110)-hIgG1 Fc (aa 245–470) Kowalczyk-Quintas et al., 2019 N/A Fixable viability dye eFluor450 eBioscience Cat# 65-0863-14 Brefeldin A Biolegend Cat# 420601 Critical commercial assays Neon Transfection System 10 mL Kit Thermo Fischer Scientific Cat# MPK1096 NEBuilder HiFi DNA Assembly Cloning Kit New England Biolabs Cat# E5520S jetPEI Polyplus transfection Cat# 101-10N Experimental models: Cell lines DG-75 DSMZ Cat# ACC 83 HEK 293T DSMZ Cat# ACC 635 Oligonucleotides 5ʹ-TCCTCCATGGCAACTACACGTGG-3ʹ Integrated DNA Technologies Hs.Cas9.CD79A.1.AB 5ʹ-AACACCTCGGAGGTCTACCAGGG-3ʹ Integrated DNA Technologies Hs.Cas9.CD79B.1.AA 5ʹ-CCTTCCAAGGACGTCATGCAGGGC-3ʹ Integrated DNA Technologies N/A 5ʹ-TTAATAACATCACCATGCACAGG-3ʹ Integrated DNA Technologies Hs.Cas9.LYN.1.AE 5ʹ-CTCACCGTCCTTGTCTCCGTCGG-3ʹ Integrated DNA Technologies N/A 5ʹ-GTTTGGAAGAGGATCACACTCGG-3ʹ Integrated DNA Technologies Hs.Cas9.CD27.1.AF 5ʹ-CAAATACACGAACTTCTCCC-3ʹ Integrated DNA Technologies Hs.Cas9.TCL1A.1.AE 5ʹ-CTCGGGAAGGTACCAAGGAT-3ʹ Integrated DNA Technologies Hs.Cas9.TNFRSF13B.1.AA Software and algorithms Flow Jo_v10 Flow Jo https://www.flowjo.com/solutions/flowjo RRID: SCR_008520 GraphPad Prism 8.4 GraphPad https://www.graphpad.com/scientific- software/prism RRID: SCR_002798 Image J V.2.3.0 Rasband, 1997-2018 http://imagej.net/ImageJ RRID: SCR_003070 ClustVis Metsalu and Vilo, 2015 https://biit.cs.ut.ee/clustvis/; RRID: SCR_017133 String Szklarczyk et al., 2020 https://www.string-db.org; RRID: SCR_005223 Cell Reports 39, 111019, June 28, 2022 e3

Techniques: Expressing, Western Blot, Control

Figure 4. BAFFR regulates the expression of PTEN and FOXO1 (A) Western blot analysis of PTEN and FOXO1 in naive and MBCs treated for 24–72 h ± BAFF (20 ng/mL). N-fold changes of PTEN and FOXO1 expression were calculated as signals normalized to actin and further normalized to untreated samples from day 0 per subset. The experiment was performed once in this format. (B) Flow cytometric analysis of PTEN expression in naive and MBCs treated for 24–72 h ± BAFF. N-fold change of PTEN MFI was calculated as [(PTEN MFI + BAFF)/(PTEN MFI –BAFF)]. (C) Western blot analysis of phosphorylated FOXO1 (S256) in naive B cells treated with BAFF or F(ab0)2 anti-IgM (2 mg/mL) for 0.5–8 h. N-fold change in pFOXO1 levels was calculated as described in (A). (D) Western blot analysis of PTEN in naive B cells treated with inhibitors against SYK (2.5 mM R406), PI3Kd (0.625 mM nemiralisib), PDK1 (5 mM BX-795), NIK (10 mM SMI1), or DMSO (control) ± BAFF for 2 days. (E) Western blot analysis of FOXO1 in naive B cells treated with iSYK, iNIK, or DMSO ± BAFF for 2 days. (D and E) Blots are representative of R2 independent experiments. N-fold changes were calculated as signals normalized to actin and further normalized to the control untreated sample. (F) Flow cytometric analysis of CD62L and CXCR4 in naive and MBCs treated overnight with BAFF in the presence of FOXO1 inhibitor (2.5 mM AS1842856) or DMSO (control). Plots show the mean of CD62L and CXCR4 MFI from 2 independent experiments (2–3 replicates/experiment, 2 HDs). *p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant (2-way ANOVA with Tukey multiple comparisons test).

Journal: Psychiatry research

Article Title: Genetic variations in evolutionary accelerated regions disrupt cognition in schizophrenia.

doi: 10.1016/j.psychres.2022.114586

Figure Lengend Snippet: Figure 4. BAFFR regulates the expression of PTEN and FOXO1 (A) Western blot analysis of PTEN and FOXO1 in naive and MBCs treated for 24–72 h ± BAFF (20 ng/mL). N-fold changes of PTEN and FOXO1 expression were calculated as signals normalized to actin and further normalized to untreated samples from day 0 per subset. The experiment was performed once in this format. (B) Flow cytometric analysis of PTEN expression in naive and MBCs treated for 24–72 h ± BAFF. N-fold change of PTEN MFI was calculated as [(PTEN MFI + BAFF)/(PTEN MFI –BAFF)]. (C) Western blot analysis of phosphorylated FOXO1 (S256) in naive B cells treated with BAFF or F(ab0)2 anti-IgM (2 mg/mL) for 0.5–8 h. N-fold change in pFOXO1 levels was calculated as described in (A). (D) Western blot analysis of PTEN in naive B cells treated with inhibitors against SYK (2.5 mM R406), PI3Kd (0.625 mM nemiralisib), PDK1 (5 mM BX-795), NIK (10 mM SMI1), or DMSO (control) ± BAFF for 2 days. (E) Western blot analysis of FOXO1 in naive B cells treated with iSYK, iNIK, or DMSO ± BAFF for 2 days. (D and E) Blots are representative of R2 independent experiments. N-fold changes were calculated as signals normalized to actin and further normalized to the control untreated sample. (F) Flow cytometric analysis of CD62L and CXCR4 in naive and MBCs treated overnight with BAFF in the presence of FOXO1 inhibitor (2.5 mM AS1842856) or DMSO (control). Plots show the mean of CD62L and CXCR4 MFI from 2 independent experiments (2–3 replicates/experiment, 2 HDs). *p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant (2-way ANOVA with Tukey multiple comparisons test).

Article Snippet: Chemicals, peptides, and recombinant proteins BAFF Smulski et al., 2017 N/A CD40L Smulski et al., 2017 N/A SARS-Cov2 spike protein Thermofisher Cat# RP-87680 SYK inhibitor (R406) Selleckchem Cat# S2194; CAS 841290-81-1 Dual BCR-ABL/LYN inhibitor (Bafetinib/INNO-406) Selleckchem Cat# S1369; CAS 859212-16-1 PI3Kd inhibitor (nemiralisib/GSK2269557) Selleckchem Cat# S7937; CAS 1254036-71-9 PDK1 inhibitor (BX-795) Selleckchem Cat# S1274; CAS 702675-74-9 FOXO1 inhibitor (AS1842856) Selleckchem Cat# S8222; CAS 836620-48-5 NIK inhibitor (SMI1) Hycultec Cat# HY112433; CAS 1660114-31-7 human monoclonal IgG1l anti-BAFF antibody belimumab (Benlysta) GlaxoSmithKline Pharmaceuticals CAS 356547-88-1 hTACI (aa 31–110)-hIgG1 Fc (aa 245–470) Kowalczyk-Quintas et al., 2019 N/A Fixable viability dye eFluor450 eBioscience Cat# 65-0863-14 Brefeldin A Biolegend Cat# 420601 Critical commercial assays Neon Transfection System 10 mL Kit Thermo Fischer Scientific Cat# MPK1096 NEBuilder HiFi DNA Assembly Cloning Kit New England Biolabs Cat# E5520S jetPEI Polyplus transfection Cat# 101-10N Experimental models: Cell lines DG-75 DSMZ Cat# ACC 83 HEK 293T DSMZ Cat# ACC 635 Oligonucleotides 5ʹ-TCCTCCATGGCAACTACACGTGG-3ʹ Integrated DNA Technologies Hs.Cas9.CD79A.1.AB 5ʹ-AACACCTCGGAGGTCTACCAGGG-3ʹ Integrated DNA Technologies Hs.Cas9.CD79B.1.AA 5ʹ-CCTTCCAAGGACGTCATGCAGGGC-3ʹ Integrated DNA Technologies N/A 5ʹ-TTAATAACATCACCATGCACAGG-3ʹ Integrated DNA Technologies Hs.Cas9.LYN.1.AE 5ʹ-CTCACCGTCCTTGTCTCCGTCGG-3ʹ Integrated DNA Technologies N/A 5ʹ-GTTTGGAAGAGGATCACACTCGG-3ʹ Integrated DNA Technologies Hs.Cas9.CD27.1.AF 5ʹ-CAAATACACGAACTTCTCCC-3ʹ Integrated DNA Technologies Hs.Cas9.TCL1A.1.AE 5ʹ-CTCGGGAAGGTACCAAGGAT-3ʹ Integrated DNA Technologies Hs.Cas9.TNFRSF13B.1.AA Software and algorithms Flow Jo_v10 Flow Jo https://www.flowjo.com/solutions/flowjo RRID: SCR_008520 GraphPad Prism 8.4 GraphPad https://www.graphpad.com/scientific- software/prism RRID: SCR_002798 Image J V.2.3.0 Rasband, 1997-2018 http://imagej.net/ImageJ RRID: SCR_003070 ClustVis Metsalu and Vilo, 2015 https://biit.cs.ut.ee/clustvis/; RRID: SCR_017133 String Szklarczyk et al., 2020 https://www.string-db.org; RRID: SCR_005223 Cell Reports 39, 111019, June 28, 2022 e3

Techniques: Expressing, Western Blot, Control