Journal: Journal of Human Immunity

Article Title: Recurrent severe viral infection in a child with inherited complete TBK1 deficiency

doi: 10.70962/jhi.20250058

Figure Lengend Snippet: Homozygosity for a LOF TBK1 mutation in the patient. (A) Family pedigree showing segregation of the TBK1 mutation. PCR products were amplified from genomic DNA extracted from the granulocytes of P1 and both parents and subjected to Sanger sequencing. (B) TBK1 mRNA levels (upper panel) were determined by RT-qPCR on HEK293T cells 24 and 48 h after transfection with empty vector (EV), WT, and mutant TBK1 constructs. Western blot analysis was performed to assess the levels of protein for TBK1 (lower panel), autophosphorylated TBK1 (p-TBK1, Ser172), IRF3, and autophosphorylated IRF3 (p-IRF3, Ser396) in HEK293T cells at 24 and 48 h after transfection with EV, N-terminally flag-tagged WT, and mutant TBK1 constructs. The results shown are representative of three independent experiments. (C) Pulse-chase analysis of WT and mutant TBK1 protein stability. HEK293T cells were transfected with flag-tagged WT or mutant TBK1 expression plasmids for 24 h. Cells were then treated with cycloheximide (CHX; 100 ng/ml) for the indicated time points to inhibit protein synthesis, followed by western blot analysis (bottom). TBK1 protein levels were quantified by densitometry, normalized to GAPDH, and plotted over time (top). Data shown are representative of three independent experiments. The data shown are the means ± SEM of three independent experiments. P values were obtained by one-way ANOVA with Tukey’s multiple comparisons tests, and the P values for the 4-, 8-, and 12-h time points are indicated for the comparison of P1’s cells with control cells. ns; P > 0.05; *P < 0.05; and **P < 0.01. (D) ISRE, IRF3, and NF-κB promoter-driven luciferase reporter assays were performed on HEK293T cells 24 h after transfection with ISRE, IRF3, or NF-κB reporter plasmids along with EV, WT, and mutant TBK1 constructs. Luciferase activity was measured to assess TBK1-mediated activation. The results shown are representative of three independent experiments. The data shown are the means ± SEM of three experiments with three biological replicates, P values were obtained by one-way ANOVA with Tukey’s multiple comparisons tests. ns; P > 0.05, ****P < 0.0001. Source data are available for this figure: .

Article Snippet: Primary antibodies against the following proteins were used in this study: TBK1 (N-ter) (ab40676; Abcam), TBK1 (C-ter) (#3504; Cell Signaling Technology), p-TBK1 (Ser172) (#5483; Cell Signaling Technology), IRF3 (66670-1-Ig; Proteintech), p-IRF3 (Ser396) (#4947; Cell Signaling Technology), Flag (A8592; Sigma-Aldrich).

Techniques: Mutagenesis, Amplification, Sequencing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Construct, Western Blot, Pulse Chase, Expressing, Comparison, Control, Luciferase, Activity Assay, Activation Assay

Journal: Journal of Human Immunity

Article Title: Recurrent severe viral infection in a child with inherited complete TBK1 deficiency

doi: 10.70962/jhi.20250058

Figure Lengend Snippet: Impaired induction of IFNs via TBK1-mediated pathways in the patient’s SV40-fibroblasts. (A) TBK1 mRNA levels (left panel) were measured by RT-qPCR in fibroblasts (SV40-fibroblasts) from healthy controls (C1, C2), P1, and two other TBK1-deficient patients (WT/G159A, W619*/W619*), and an immunoblot analysis of endogenous TBK1 protein levels (right panel) was performed with antibodies against the N-terminus and C-terminus of TBK1. The results shown are representative of three independent experiments. (B) SV40-fibroblasts from healthy controls (C1, C2), P1, two other TBK1 patients (WT/G159A, W619*/W619*), and a TLR3 −/− HSE patient were left unstimulated (NS) or were stimulated with poly (I:C) alone, Lipofectamine alone (Lipo), or both (poly (I:C)+Lipo), for 6 h. The relative expression levels of IFNB1 , IFNL1 , IFIT1 , and IL6 were measured by RT-qPCR. The results shown are from three independent experiments. (C) IFNB mRNA levels were measured by RT-qPCR in SV40-F from healthy controls ( n = 3), P1, and HSE patients with TBK1-deficient (WT/G159A) and AR IFNAR1 deficiencies. Cells were either left uninfected (NS) or infected with HSV-1 (KOS strain, MOI = 1) for 24 h. Data represent the means of three independent experiments. (D) SV40-F from healthy controls (Ctrls, n = 3), P1, and HSE patients with TBK1-deficient (WT/G159A) and AR IFNAR1 deficiencies were either left untreated or pretreated with IFN-β for 24 h, followed by infection with HSV-1 (MOI = 0.001). Viral replication was assessed at the indicated time points post-infection using the TCID 50 virus titration method. Data represent means ± SEM from three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test; P values at 48 h after infection compare P1’s cells to controls. ***P < 0.0001. Source data are available for this figure: .

Article Snippet: Primary antibodies against the following proteins were used in this study: TBK1 (N-ter) (ab40676; Abcam), TBK1 (C-ter) (#3504; Cell Signaling Technology), p-TBK1 (Ser172) (#5483; Cell Signaling Technology), IRF3 (66670-1-Ig; Proteintech), p-IRF3 (Ser396) (#4947; Cell Signaling Technology), Flag (A8592; Sigma-Aldrich).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Infection, Virus, Titration

Journal: Biomedicines

Article Title: Functional Disruption of IQGAP1 by Truncated PALB2 in Two Cases of Breast Cancer: Implications for Proliferation and Invasion

doi: 10.3390/biomedicines13081804

Figure Lengend Snippet: Key domains and interaction regions of PALB2 protein and the P-T1 and P-T2 variants. ( A ) Domains and interaction regions of the PALB2 protein. The upper circles show the molecular surface representation of the Coiled–coil PALB2 (orange) dimerization, coil–coil PALB2-BRCA1 (green) interaction, PALB2-MRG15 (yellow) interaction, and PALB2-BRCA2 (cyan) interaction. The lower circles show the intrinsically disordered structure of the N-terminal (AlphaFold 2 prediction) and the β-sheet structure of the WD40 domain at the C-terminal. Ab1 and Ab2 represent the antigenic regions recognized by the PA5-48258 and BS-0588R PALB2 antibodies, respectively. ( B , C ) Diagram of the domains and interaction regions of PALB2 variants P-T1 and P-T2, respectively. CC, coil–coil motif; ChAM, chromatin association motif; FX, FXLP motif; NLS, nuclear export signal; BRCA1, BRCA1 DNA repair-associated; KEAP1, Kelch-like ECH-associated protein 1; RAD51, RAD51 recombinase; MRG15, MORF-related gene 15; BRCA2, BRCA2 DNA repair-associated; RAD51C, RAD51 paralog C; XRCC3, X-ray repair cross complementing 3; POLE, DNA polymerase epsilon (catalytic subunit) and RNF168, ring finger protein 168.

Article Snippet: PALB2 (C-ter) , , Rabbit polyclonal , , 1:200 , Bioss 2 , BS-0588R.

Techniques:

Journal: Biomedicines

Article Title: Functional Disruption of IQGAP1 by Truncated PALB2 in Two Cases of Breast Cancer: Implications for Proliferation and Invasion

doi: 10.3390/biomedicines13081804

Figure Lengend Snippet: PALB2 full-length protein. Western blot of isolated proteins from HeLa cells and from circulating white cells from control IDC and cases with truncated variants of PALB2, P-T1, and P-T2 probed with PALB2 C-terminus antibody.

Article Snippet: PALB2 (C-ter) , , Rabbit polyclonal , , 1:200 , Bioss 2 , BS-0588R.

Techniques: Western Blot, Isolation, Control

Journal: Biomedicines

Article Title: Functional Disruption of IQGAP1 by Truncated PALB2 in Two Cases of Breast Cancer: Implications for Proliferation and Invasion

doi: 10.3390/biomedicines13081804

Figure Lengend Snippet: PALB2 (green) and IQGAP1 (red) expression in breast cancer control (CC1 and in CC2) mutated breast cancer (P-T1) labeled with antibodies binding to N-terminal or C-terminal domains. In CC2, PALB2-specific fluorescence at high intensity in cytosol and to a lesser extent in the nuclei of tumor cells (white arrowheads); IQGAP1-specific immunofluorescence in the plasma membrane and cytosol of most cells (yellow arrows) at two different intensities, high and medium level, in a dot-like pattern distribution; in the PALB2-IQGAP1 merged image, these two levels are more evident, and colocalization exists to a certain extent. B-T1 anti-PALB2-N-terminus antibody immunofluorescence signal present in both cytosol (high) and nucleus (medium and homogeneous); IQGAP1-specific immunofluorescence is displayed at a homogeneous medium intensity level in the plasma and cytosol of most cells. C-terminus anti-PALB2 antibody in CC1 samples exhibited similar immunolabeling to the N-terminus (white arrowheads). The IQGAP1 localization pattern of two intensity levels and localization is also shown in the first lane of the images. B-T1 and C-terminus anti-PALB2 antibody fluorescence at medium–high intensity, mainly in cytosol and at much lower intensity in the nuclei of cancer cells (white arrowheads). IQGAP-specific immunofluorescence at medium- to high-level intensity in cytosol and plasma membrane, frequently colocalizing (merged) in some tumor areas (yellow arrows). Scale bar = 40 µm.

Article Snippet: PALB2 (C-ter) , , Rabbit polyclonal , , 1:200 , Bioss 2 , BS-0588R.

Techniques: Expressing, Control, Labeling, Binding Assay, Fluorescence, Immunofluorescence, Clinical Proteomics, Membrane, Immunolabeling

Journal: Biomedicines

Article Title: Functional Disruption of IQGAP1 by Truncated PALB2 in Two Cases of Breast Cancer: Implications for Proliferation and Invasion

doi: 10.3390/biomedicines13081804

Figure Lengend Snippet: Diagrams of PALB2 and IQGAP1 expression in truncated PALB2 human breast invasive ductal carcinoma. AU—arbitrary units. ( A ) Specific PALB2 expression in nucleus or cytosol in control (CC) IDC and in truncated PALB2 (P-T) IDC samples probed with antibodies against N- or C-terminus of PALB2 protein (* p < 0.05). ( B ) IQGAP1 total expression and peri-plasma membrane expression ( p < 0.05). ( C ) In subpopulations of cells (25–30% of total cells), correlation between the expression of IQGAP1 and PCNA evidenced that high expression of PCNA corresponded to lower expression of IQGAP1, which was higher in truncated PALB2 cases ( p < 0.05). ( D ) Correlation of IQGAP1/CK7 expression-level quotients in control and in PALB2-truncated cases. The correlation is lower in PALB2-truncated cases ( p > 0.05). In ( C , D ), point clouds are not represented to improve the clarity of the plots. ( E ) The Pearson correlation coefficient (PCC) was calculated for each sample to quantify the degree of colocalization between fluorophores.

Article Snippet: PALB2 (C-ter) , , Rabbit polyclonal , , 1:200 , Bioss 2 , BS-0588R.

Techniques: Expressing, Control, Clinical Proteomics, Membrane