Journal: bioRxiv

Article Title: Bardet-Biedl Syndrome 1 Mutations Differentially Impact BBSome Integrity and its Function in Ciliary Trafficking

doi: 10.64898/2026.01.23.701255

Figure Lengend Snippet: Representative micrographs depicting BBS9 localisation (A) and quantification of ciliary BBS9 (B) in the parental and BBS1 variants expressing WT and BBS1 KO RPE1 cell lines upon 24 h serum starvation. Antibody against acetylated tubulin (Ac-tub) was used to stain the primary cilia. Nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 5µm. Analysis was carried out using the Fiji ImageJ. Mean and SD from three independent experiments (n = 160-230 cilia). (C) Distribution of BBS9 signal along the axoneme in BBS1 KO RPE1 cells expressing BBS1 WT and R160Q variants in (A). Quantification was performed using the Fiji ImageJ. The plot shows mean and SD from n = 41 cilia for WT and n = 35 cilia for the R160Q variant. (D) Co-immunoprecipitation of the BBSome subunits from mCherry-FLAG tagged BBS1 variants expressing WT and BBS1 KO RPE1 cell lines using anti-FLAG antibodies after 24 h serum starvation. Representative immunoblot out of three experiments is shown. (E) Quantification of the co-precipitated BBSome subunits in (D) was performed using the Fiji ImageJ software. Amount of the BBSome subunits was normalized to the levels of the mCherry-FLAG tagged BBS1 WT in BBS1 KO RPE1 cell line detected on the respective membrane. Mean with SD of three experiments is shown.

Article Snippet: Membranes were developed using chemiluminescence immunoblot imaging system Fusion Solo S (Vilber).

Techniques: Expressing, Staining, Variant Assay, Immunoprecipitation, Western Blot, Software, Membrane

Journal: bioRxiv

Article Title: Bardet-Biedl Syndrome 1 Mutations Differentially Impact BBSome Integrity and its Function in Ciliary Trafficking

doi: 10.64898/2026.01.23.701255

Figure Lengend Snippet: Representative micrographs (A) and quantification (B) showing localisation of BBS9 in pericentriolar satellites in the parental and BBS1 variants expressing WT and BBS1 KO RPE1 cell lines. Antibody against PCM-1 was used to stain the satellites. Nucleus was stained with 4′,6-diamidino-2- phenylindole (DAPI). Scale bar, 5µm. Analysis was carried out using the Fiji ImageJ software. Mean and SD from three independent experiments (n = 220-340 satellites). Statistical significance was calculated using two-tailed paired t-test. (C) Co-immunoprecipitation of the BBSome subunits from YFP-BBS4 and BBS1 variants expressing BBS1 KO RPE1 cell lines using anti-GFP antibodies after 24 h serum starvation. Representative immunoblot out of three experiments is shown. Quantification of the co-precipitated BBS8 and BBS1 (D) and BBS5 and BBSome core subunits (E) in (C) was performed using the Fiji ImageJ. Amount of the BBSome subunits was normalized to the YFP-BBS4 levels in the mCherry-FLAG tagged BBS1 WT expressing BBS1 KO RPE1 cell line, as detected on the respective membrane. Mean with SD of three experiments is shown.

Article Snippet: Membranes were developed using chemiluminescence immunoblot imaging system Fusion Solo S (Vilber).

Techniques: Expressing, Staining, Software, Two Tailed Test, Immunoprecipitation, Western Blot, Membrane

Journal: bioRxiv

Article Title: First inhibitor of a bacterial two-partner secretion system

doi: 10.64898/2026.01.11.698920

Figure Lengend Snippet: ( A ) P1 CPP prevented secretion of FHA and ACT from B. bronchiseptica RB50. RB50 was grown in non-virulence factor repressive conditions before transfer to virulence inducing conditions and treatment with P1 CPP (10 μM) for 1h (see Methods ), n = 3. Proteins released to culture media were probed by immunoblotting with either ⍺FHA or ⍺ACT antisera. ( B ) P1 CPP inhibited secretion of FHA from B. pertussis clinical isolates. Strains L1423, L1756, and L2228 were grown similarly to A except bacteria were treated with P1 CPP (20 μM) for 2h. Proteins released to culture media and whole cell lysates were probed by immunoblotting with ⍺FHA. ( C ) P1 CPP treatment reduces virulence factor release from B. pertussis . Volcano plot of LC-MS/MS data showing differential protein levels in the supernatant (n = 3). Red symbols indicate proteins of significantly lower abundance in the culture media of P1 CPP treated cells. Green symbols indicate proteins of significantly higher abundance. Vertical lines indicate fold-change cutoff >20%. Horizontal line indicates q-value cutoff at 0.05. Full proteomics datasets are in Table S7 ( D ) P1 E15R - CPP prevents B. bronchiseptica adherence to lung cells. Adherent RB50 infection of A549 cell line in the presence of inhibitor treatment (or mock treatment) quantified by recovered colony count, n = 3. Isogenic RB50 ΔfhaB and RB50 ΔfhaC strains were included as controls. ( E ) P1 E15R - CPP prevents B. pertussis adherence to lung cells and macrophages. Adherent L1423 after infection of either A549 lung or macrophage differentiated THP-1 cell lines in the presence of inhibitor treatment (or mock treatment) quantified by microscopy, n = 3. L1423 bacteria were transformed with a plasmid expressing fuGFP. ( F ) P1 E15R - CPP prevents B. pertussis induced cytotoxicity and adherence. Representative micrographs from infected A549 cells in E are shown. DNA was labelled with DAPI, and α-tubulin was labelled with a corresponding antiserum. Sample micrographs of treated infected THP-1 cells are in Figure S8. ANOVA statistics for all panels in Table S8.

Article Snippet: Membranes were blocked overnight with immunoblotting buffer [1:1 Intercept Blocking Buffer (Li-Cor):PBS + 0.01% Tween-20], incubated overnight with 1° antibody diluted in immunoblotting buffer (αHis or αStrep at 1:5,000 dilution; αBamA, αDegP, or αSurA at 1:10,000 dilution), washed with PBS-T (PBS + 0.01% Tween-20), incubated with 2° antibody (1:5,000 dilution) for 2 h, and finally washed two times with PBS-T and three times with PBS.

Techniques: Western Blot, Bacteria, Liquid Chromatography with Mass Spectroscopy, Infection, Microscopy, Transformation Assay, Plasmid Preparation, Expressing

Journal: Frontiers in Molecular Neuroscience

Article Title: Interactome screening implicates BAG6 as a suppressor of UBQLN2 misfolding in ALS/FTD

doi: 10.3389/fnmol.2025.1720347

Figure Lengend Snippet: UBQLN2 4XALS exhibits enhanced association with PEG10 but retains degradation activity. (A) co-IP of UBQLN2 and PEG10 from iPSCs expressing UBQLN2 WT , UBQLN2 P497H , or UBQLN2 4XALS with or without BAG6 knockdown. Immunoblotting with α-PEG10 antibodies revealed increased UBQLN2–PEG10 interaction in UBQLN2 P497H and UBQLN2 4XALS backgrounds, particularly under BAG6 knockdown conditions. (B) Quantification of PEG10 co-IP across genotypes demonstrates elevated PEG10 binding to UBQLN2 P497H and UBQLN2 4XALS compared to UBQLN2 WT . Statistical analysis of (A) was performed using two-way ANOVA with Tukey’s post hoc test. Data are presented as mean ± SEM, n = 3; ∗p ≤ 0.05, ∗∗ p ≤ 0.01. (C) Reciprocal co-IP of UBQLN2 using α-PEG10 antibodies in UBQLN2 WT , UBQLN2 P497H , UBQLN2 4XALS , or UBQLN2 I498X iPSCs with or without BAG6 knockdown. (D) Quantification of UBQLN2 co-IP results from panel C demonstrated elevated PEG10 binding to UBQLN2 P497H and UBQLN2 4XALS compared to UBQLN2 WT under conditions of BAG6 knockdown. Statistical analysis of (C) was performed using two-way ANOVA with Tukey’s post hoc test. Data are presented as mean ± SEM, n = 3; ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.005. (E) Quantification of PEG10 RF1/2 levels from input lysates in (C) , normalized to total PEG10 (RF1/2 + RF1). Data were analyzed by two-way ANOVA with Tukey’s post hoc test. Values represent mean ± SEM, n = 3; ∗∗∗∗ p ≤ 0.0001.

Article Snippet: The following secondary antibodies were used for immunoblotting: IRDye 680RD goat anti-mouse (926–68,070) and IRDye 800CW goat anti-rabbit (926–32,211), both from LI-COR.

Techniques: Activity Assay, Co-Immunoprecipitation Assay, Expressing, Knockdown, Western Blot, Binding Assay