Fig 2: Knockdown of SLPI increases phosphorylated NF-?B p105 protein expression in PDL cells. The cells were transfected with either SLPI siRNA or NC construct for 96 h and treated with or without 1 µg/ml LPS during the last 30 min of the 96 h transfection-period. The I?Ba (a) and the phosphorylated NF-?B p105 (b) and p65 (c) proteins were determined in PDL cell lysates by Western blotting at their respective expected molecular weights. I?Ba is detected as one band at 37 kDa, phosphorylated NF-?B p105 as one band just over 100 kDa and phosphorylated NF-?B p65 as one band at 65 kDa. The relative I?Ba and phosphorylated NF-?B p105 and p65 concentrations were assessed by densitometry and normalized to GAPDH serving as internal control. Values are presented as mean ± SEM of 3 observations in each group. * and ** represent P < 0.05 and P < 0.01, respectively, versus NC ctrl. ns non-significant

Fig 3: NFkB1 expression in and nuclear translocation in transfected HEK293 cells. Total proteins were extracted from transfected HEK293 cells to determine the expression of p105/50. (A) For microscopic analysis of NFkB1-p50 translocation into the nucleus, HEK293 cells transiently expressing the eGFP-p50 wild type and mutant were used. Nuclei were stained with DAPI. The wild-type p50-construct localizes into the nucleus while the mutant p50-construct was retained in the cytoplasm. (B) Western blot to analyze the effect of the variant on the expression of p105/50 in the transfected HEK293 cells. The expression in the wild type was prominent in the wild type, while no noticeable expression in the variant was observed. (C) p50 expression in HEK293 showed an increase in wild-type and a decrease in the mutant (p. R230C) in the nuclear extract.

Fig 4: Monoallelic NFKB1 missense mutation in a family with late-onset antibody deficiency. (A) Segregation of NFKB1variant was analyzed by sequencing genomic PCR product and revealed an autosomal-dominant inheritance in families with reduced clinical penetrance. The analysis excluded the mother of patients because of material lack. (B) Sanger sequencing of genomic PCR products results in the chromatogram of missense variant and wild type (WT) (C) Structure of NF?B protein showing the position of the identified mutation. (D) Immunoblotting was performed in PBMCs of subjects (S1, S2, and S3) and healthy control (HC), and the expression of p105/50 was evaluated. The expression of p105 was reduced for all the subjects compared to the control. However, the expression of p50 was reduced in S2. (E) PBMCs from HC and S1, S2 and S3 were stimulated with PMA; 50 ng/ml and ionomycin; 1 µg/ml and the expression of p105/50 evaluated. There were no significant changes in the p105/50 expression after stimulation in the subjects; however, p105 phosphorylation at serine 933 was detected in only the HC but not in the subject. Beta-actin was used as a cytoplasmic loading control.

Fig 5: c-Myc accumulation in outlet obstruction.(A) Fold change (vs. sham) of c-Myc (Myc), Hdac3 and Ezh2 mRNAs in outlet obstruction and de-obstruction (n=6-8). (B) Inverse correlation between miR-29b and Myc mRNA level (both expressed relative to sham) in outlet obstruction and its reversal (n=6-8). The leftmost symbol represents 10 d obstruction, the rightmost symbol represents de-obstruction, and the symbols close to x=1 represent sham and 6 weeks of obstruction, respectively. Western blots and bar graph summaries showing increased c-Myc (C) and Ezh2 (D) levels in outlet obstruction (n=6 for both). (E) Western blots and bar graph summary showing increased phospho-NF-?B p105 in outlet obstruction (n=6). Blots for phospho-NF-?B p65 and I?B-a are shown without bar graph summaries as no significant differences were seen.