Journal: Stem Cells International

Article Title: Electroacupuncture Improved Hippocampal Neurogenesis following Traumatic Brain Injury in Mice through Inhibition of TLR4 Signaling Pathway

doi: 10.1155/2017/5841814

Figure Lengend Snippet: EA treatment exerted an inhibitory effect on both of the two downstream pathways of TLR4 in the hippocampus posttrauma. Hippocampal TLR4, Myd88, TRAF6, TRAM, TRIF, and NF- κ B expression of the sham, TBI, TBI + EA, TBI + EA + LPS, TBI + EA + Veh, and sham + EA groups ( n = 3 in each group) were detected by WB at 35 days after TBI. (a–f) The six kinds of proteins in TLR4/Myd88 κ B and TLR4/TRIF/ κ B signaling pathways were upregualted in the TBI group compared with the sham group and the sham + EA group. EA treatment resulted in a significant decrease in the TBI + EA group, but the effects were attenuated by LPS administration in the TBI + EA + LPS group. No difference of the proteins between the TBI + EA and TBI + EA + Veh groups was observed. ∗ P < 0.05 versus the TBI group, † P < 0.05 versus the TBI + EA + LPS group.

Article Snippet: Membranes were blocked with 5% nonfat milk solution in tris-buffered saline with 0.1% Triton X-100 (TBST) for 1 hour and then incubated overnight at 4°C with appropriate primary antibodies as below: rabbit anti-mouse TLR4 antibody (1 : 1000, Thermo Fisher, PA5-23125, Rockford, IL, USA), rabbit anti-mouse myeloid differentiation factor 88 (Myd88) antibody (1 : 500, Santa Cruz, sc-17320, Dallas, TX, USA), rabbit anti-mouse TNFR-associated factor (TRAF6) antibody (1 : 1000, Novus Biological, NB100-56179, Littleton, CO, USA), rabbit anti-mouse toll/IL-1 receptor domain-containing adapter-induced interferon- β (TRIF) antibody (1 : 1000, Enzo Life Sciences, ALX-215-016, Farmingdale, NY, USA), rabbit anti-mouse TRIF-related adaptor molecule (TRAM) antibody (1 : 1000, OriGene, TA-306163, Rockville, MD, USA), rabbit anti-mouse nuclear factor- κ B (NF- κ B) p65 antibody (1 : 1000, GeneTex, GTX21893, Irvine, CA, USA), and rabbit anti- β -actin antibody (1 : 2000, Proteintech, 20536-1-AP, Rosemont, IL, USA).

Techniques: Expressing

Journal: Scientific Reports

Article Title: Lung cancer exosomal Gal3BP promotes osteoclastogenesis with potential connotation in osteolytic metastasis

doi: 10.1038/s41598-024-79006-w

Figure Lengend Snippet: Fig. 6. Mechanistic assessment of OCs in LC-OC interaction. Differentiated OCs alone or co-incubated with LC cells were lysed after the second treatment of RANKL (day 4) to assess any changes in the expression levels of critical markers via western blot. The immunoblots were analyzed using ImageJ and plotted for the following markers: (a) NFAT2, (b) TRAF6, (c-d) p-44/42 MAPK, and p-SAPK/JNK using GAPDH as loading control with (e-f) representative blots shown. Each data point represents mean ± SD (n = 4); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. RANKL.

Article Snippet: The membrane was blocked with 5% BSA (Fisher Scientific, Pittsburgh, PA), washed (1xTBST) and probed with the following antibodies overnight at 4 °C: anti-p42/44 MAPK (42, 44 kDa, 1:1000), anti- p-SAPK/JNK (46, 54 kDa, 1:1000), anti-CD9 (22, 24, 35 kDa, 1:1000), anti-NFAT2 (140 kDa, 1:1000), anti-CD40/TRAP (31 kDa, 1:1000) (Abcam, Cambridge, United Kingdom) anti-TRAF6 (60 kDa 1:1000), anti-cFos (62 kDa, 1:1000) and DC-Stamp (100 kDa, 1:500, Novus Biologics, Centennial, CO).

Techniques: Incubation, Expressing, Western Blot, Control

Journal: Nucleic Acids Research

Article Title: TRAF6 mediates human DNA2 polyubiquitination and nuclear localization to maintain nuclear genome integrity

doi: 10.1093/nar/gkz537

Figure Lengend Snippet: hTRAF6 promotes hDNA2 nuclear localization. ( A ) HeLa cells were transfected with an empty control vector (EV) or a vector encoding myc-hTRAF6. Endogenous hDNA2 protein in the CE and NE was analyzed by western blot analysis using an anti-hDNA2 antibody. GAPDH and histone H3 were used as CE and NE markers, respectively. ( B ) 3xFlag-hDNA2 was co-expressed with EV or a vector encoding WT or C70A (ubiquitin E3 ligase activity-deficient) mutant hTRAF6 in 293T cells. 3xFlag-hDNA2 in the CE and NE was analyzed by western blot analysis using an anti-Flag antibody. GAPDH and histone H3 were used as CE and NE markers, respectively. ( C – E ) 293T cells co-expressing 3xFlag-hDNA2 and myc-hTRAF6 were left untreated or were treated with ( C ) a small molecule inhibitor of TRAF6 E3 ligase activity (C25–140, 30 μM, 48 h), ( D ) a TRAF6/RANK interaction-disrupting peptide (T6DP, 100 μM, 24 h) or ( E ) a small molecule ATM kinase activity inhibitor (KU-55933, 10 μM, 16 h). 3xFlag-hDNA2 levels in the CE and NE were analyzed by western blot analysis using an anti-Flag antibody. GAPDH and histone H3 were used as CE and NE markers, respectively. ( F ) 293T cells expressing 3xFlag-hDNA2 were pre-treated with DMSO (control) or KU-55933 (10 μM, 16 h). The cells were left untreated or were treated with CPT (1 μM, 4 h). 3xFlag-hDNA2 levels in the CE and NE were analyzed by western blot analysis using an anti-Flag antibody. GAPDH and histone H3 were used as CE and NE markers, respectively.

Article Snippet: The TRAF6 inhibitory peptide T6PD (Novus Biologicals, Catalog #NBP2–26506) was added for 24 h at a final concentration of 100 μM.

Techniques: Transfection, Control, Plasmid Preparation, Western Blot, Ubiquitin Proteomics, Activity Assay, Mutagenesis, Expressing

Journal: Nucleic Acids Research

Article Title: TRAF6 mediates human DNA2 polyubiquitination and nuclear localization to maintain nuclear genome integrity

doi: 10.1093/nar/gkz537

Figure Lengend Snippet: Inhibition of hTRAF6-mediated hDNA2 ubiquitination impairs DNA end resection and HDR (See also ). ( A – D ) Immunofluorescence staining for pRPA (red) was conducted using an anti-pRPA (S33) antibody. Nuclei were stained with DAPI (blue). (A) Representative images of HeLa cells transfected with control shRNA (shControl) or shRNA against hTRAF6 (shTRAF6), followed by CPT treatment (1 μM, 4 h). (B) Relative pRPA levels in the control and TRAF6 knockdown cells treated with DMSO or CPT. (C) Relative pRPA levels in HeLa cells pretreated with DMSO or C25–140 (30 μM, 48 h) followed by treatment with DMSO or CPT (1 μM, 4 h). Representative images are shown in . (D) Relative pRPA levels in HeLa cells that were pre-treated with a cell-permeable control peptide (CP) or T6PD peptide (100 μM, 24 h), followed by treatment with DMSO or CPT (1 μM, 4 h). Representative images are shown in . pRPA intensity was quantified using ImageJ software. The relative pRPA intensity in the control cells (B: shControl/DMSO; C: DMSO/DMSO; D: CP/DMSO) was arbitrarily set to 1. All data are expressed as mean ± SEM of at least three independent assays. The P -values were calculated using two-tailed Student's t -tests. ( E – J ) CRISPR/Cas9 D10A-based DR-GFP HDR assay. 293T cells harboring the cassette for the DR-GFP reporter HDR assay were pretreated with DMSO (48 h), C5 (50 μM, 24 h) or C25–140 (30 μM, 48 h). The plasmid encoding CRISPR/Cas9 D10A nickase and a pair of sgRNAs was transfected into the 293T cells to induce DSB via double-nicking. HDR efficiency was analyzed using flow cytometry. (E–I) Representative flow cytometry plots for parental 293T cells or 293T DR-GFP cells under various conditions, as indicated in each panel. Counts within the yellow frame were scored as GFP-positive cells. (J) Percentages of GFP-positive cells among 293T DR-GFP cells under the conditions shown in panels (G–I). Data are expressed as mean ± SEM of at least three independent assays. The P -values were calculated using two-tailed Student's t -tests.

Article Snippet: The TRAF6 inhibitory peptide T6PD (Novus Biologicals, Catalog #NBP2–26506) was added for 24 h at a final concentration of 100 μM.

Techniques: Inhibition, Ubiquitin Proteomics, Immunofluorescence, Staining, Transfection, Control, shRNA, Knockdown, Software, Two Tailed Test, CRISPR, Plasmid Preparation, Flow Cytometry