Fig 2: Immunoblots of JAM-B in lysates of THP-1 monocyte whole cells, nuclear and cytosolic sub-fractions of THP-1 cells with and without treatment with 10 µM Bay-11-7082 inhibitor. (A) Immunoblot of nuclear (lane 1), cytoplasmic (lane 2) and whole cell lysates (lane 3) probed with anti-JAM-B antibodies. (B) Immunoblots of ubiquitin-enriched samples of whole cell lysates prepared by immunoprecipitation probed with anti-JAM-B antibodies (left panel) and anti-ubiquitin antibody (right panel). Negative control beads denote samples obtained following the immunoprecipitation procedure with negative control beads; control lysate was ubiquitin-captured samples from unstimulated/untreated cells, or cells treated with either 10 ng/mL LPS or 10 ng/mL TNF-a are ubiquitin-captured lysates from cells treated with inflammatory stimuli (LPS or TNFa) and in samples pooled from Bay-11-7082 (10 µM)-treated control and LPS- and TNF-a-treated samples.

Fig 3: Effects of the Bay-11-7082 inhibitor on the density of JAM-B protein species in THP-1 monocyte whole cell lysates after incubation with and without inflammatory stimuli (relative to total protein on stain-free gel). (A) =230 kDa bands, (B) 124–175 kDa bands, (C) 105 kDa band, (D) 88 kDa band (E) 65 kDa band, (F) 55 kDa band (G) 45 kDa bands, (H) 38 kDa band, (I) 31 kDa band and (J) blot image showing protein band densities in the Bay-11-7082 inhibitor-treated THP-1 whole cell lysates treated with and without inflammatory stimuli. A.U, arbitrary units; * indicates p = 0.05, ** indicates p = 0.01, *** indicates p = 0.001 after Mann–Whitney U test (n = 7). All bands were normalised to the total protein on the stain-free blots.

Fig 4: Fold-changes in JAM gene expression in response to TNF-a and LPS treatments for 24 h relative to the geometric mean of YWHAZ and ACT-ß expression. All data are the relative expression of the JAMs treated with TNF-a or LPS compared to the respective non-treated control. (A) JAM-A and JAM-B expression in THP-1-differentiated macrophages. (B–D) Relative expression of JAM-A, JAM-B and JAM-C, respectively, in THP-1 monocytes. *** indicates p = 0.0001, ** indicates p = 0.01, * indicates p = 0.05. n = 9 for TNF-a treatments and n = 6 for LPS treatments. Data are mean ± SEM.

Fig 5: Effects of the Bay-11-7082 inhibitor on the abundance of JAM-B protein species in THP-1 monocyte cytosolic cell lysates after incubation with and without inflammatory stimuli (relative to total protein on stain-free gel): (A) 215–259 kDa bands, (B) 124–175 kDa bands, (C) 105 kDa band, (D) 88 kDa band, (E) 65 kDa band, (F) 55 kDa band, (G) 45 kDa band, (H) 39 kDa band, (I) 31 kDa band and (J) blot images showing protein band densities in the Bay-11-7082 inhibitor-treated THP-1 cytosolic cell lysates in the presence and absence of inflammatory stimuli. A.U, arbitrary units; * indicates p = 0.05, ** indicates p = 0.01, *** indicates p = 0.001 after Mann–Whitney U test (n = 3).