Fig 1: 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 2: The effects of the NF-κB inhibitor Bay-11-7082 (10 µM) after 24 h treatment on JAM-B subcellular localisation in THP-1-differentiated macrophages. (A–C) DAPI, JAM-B and composite image in control-treated samples; (D–F) DAPI, JAM-B and composite image in LPS-treated samples; (G–I) DAPI, JAM-B and composite image in TNF-α-treated samples. (J) JAM-B punctate nuclear localisation quantification showing significant increase in JAM-B expression in the nucleus in the control plus Bay-11-7082-treated samples. (K) JAM-B punctate nuclear localisation quantification showing significant increase in JAM-B expression in the nucleus in the LPS plus Bay-11-7082-treated samples. (L) JAM-B punctate nuclear localisation quantification showing no significant increase in JAM-B expression in the nucleus in the TNF-α plus Bay-11-7082-treated samples. *** indicates p ≤ 0.001.
Fig 3: Western blot probing for JAM-B protein in samples of recombinant JAM-B protein and THP-1 monocyte whole cell lysates. All samples were separated by SDS-PAGE under reducing conditions using DTT. (A) Recombinant protein (left panel, lane 1) run together with protein molecular weight marker (left panel, lane 2) and cell lysates from unstimulated (right panel) and LPS- (right panel) or TNFα- (right panel) stimulated THP-1-cultured cells (right panel); (B) example chromatogram demonstrating band molecular weights and grouping to aid quantification. Representative images of more than three technical replicates.
Fig 4: Fold-changes in JAM gene expression in response to TNF-α and LPS treatments for 24 h relative to the geometric mean of YWHAZ and ACT-β expression. All data are the relative expression of the JAMs treated with TNF-α 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-α treatments and n = 6 for LPS treatments. Data are mean ± SEM.
Fig 5: Immunofluorescence of DAPI, JAM-B and G130 using a mouse anti-JAM-B monoclonal antibody (SC-293496) and an anti-GM130 polyclonal antibody (PA1-077) in THP-1 monocytes (A–D) and macrophages (E–J). (A) DAPI nuclear staining in THP-1 monocytes; (B) JAM-B staining in THP-1 monocytes; (C) G130 Golgi staining in THP-1 monocytes (stains peripheral membrane component of the cis-Golgi stack marker GOLGA2); (D) composite image showing co-localisation of JAM-B and G130; (E) DAPI staining in THP-1-differentiated macrophages; (F) JAM-B staining in THP-1-differentiated macrophages showing polarised staining (dotted line), in splitting nuclei (dashed line), inside the nucleus (solid line); (G) cis-Golgi stack marker GOLGA2; (H) composite image showing DAPI, JAM-B and GOLGA2 staining. At least three technical replicates were carried out. JAM-B staining contrasts with that of JAM-A (I) (JAM-A staining) and (J) (composite JAM-A and DAPI staining).
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