Journal: PLoS ONE

Article Title: A Common Carcinogen Benzo[a]pyrene Causes Neuronal Death in Mouse via Microglial Activation

doi: 10.1371/journal.pone.0009984

Figure Lengend Snippet: Western blotting was performed with protein harvested from BV-2 cells after 3 h, 12 h and 24 h treatment with 0.2 µM and 2.0 µM B[a]P (A). PhosphoP38 MAP kinase levels were significantly elevated in 3 h post treatment groups and also in 0.2 µM treated group after 12 h. After 24 h, there was no significant difference in the phosphoP38 MAP kinase levels in either B[a]P treated group when compared to time matched control. However, it was observed that phosphoP38 MAP kinase level in 0.2 µM B[a]P treated group after 12 h, was significantly decreased as compared to that in 3 h post treatment group. Similarly, phosphoP38 MAP kinase level in 0.2 µM B[a]P treated group after 24 h, was significantly decreased as compared to that in 12 h post treatment group. PhosphoP38 MAP kinase level was also significantly reduced in 24 h post treatment 2.0 µM group when compared to 3 h and 12 h post treatment groups of the same concentration (B). Data is represented as Mean ± SD of fold changes over control from triplicate sets of experiments. * p<0.01 for all groups as compared to control; # p<0.05 for 12 h 0.2 µM group as compared to 3 h 0.2 µM group; ## p<0.01 for 24 h 0.2 µM group as compared to 12 h 0.2 µM group; δ p<0.01 for 24 h 2.0 µM group as compared to 12 h 2.0 µM group. Primary microglia were stained with anti-phosphoP38 MAP kinase and anti-CD 11b antibodies, after 3 h and 12 h B[a]P treatment. The results show that upon treatment with B[a]P there was an increased intracellular expression of phosphoP38 MAP kinase. This was also observed after 12 h treatment. Moreover, in the 12 h treated cells, the phosphoP38 MAP kinase's fluorescent signals seemed to be localized towards the nucleus (C). White arrow denotes cells in which activation is visibly prominent. Images are representative of triplicate sets. Magnification is 20×; scale bar corresponds to 50 µ.

Article Snippet: After blocking with 7% skimmed milk, the blots were incubated overnight at 4°C with primary antibodies against iNOS (Chemicon, USA), SOD-1 (Santa Cruz Biotechnology, CA, USA), TRX (AB Frontiers, Korea; a kind gift from Dr. Ellora Sen, NBRC) and phosphoP38 MAP kinase (Cell Signalling, USA) at 1∶1000 dilutions.

Techniques: Western Blot, Control, Concentration Assay, Staining, Expressing, Activation Assay

Journal: Inflammation

Article Title: DHX9 Strengthens Atherosclerosis Progression By Promoting Inflammation in Macrophages.

doi: 10.1007/s10753-023-01836-z

Figure Lengend Snippet: Fig. 3 oxLDL stimulation promotes DHX9 interaction with p65 in macrophages. a Western blotting analysis of p38, JNK, ERK signaling in mac- rophages transfected with si-DHX9 or si-nc for 48 h and later incubated with 40 μg/mL oxLDL for 24 h. b Western blotting analysis of NF-κB signaling in macrophages transfected with si-DHX9 or si-nc for 48 h and later incubated with 40 μg/mL oxLDL for 24 h. c Co-IP detection of the interaction between DHX9 and p65 in macrophages treated with or without 40 μg/mL oxLDL for 24 h by using DHX9 antibody. d Immunofluores- cence analysis of DHX9 and P65 expression in macrophages treated with or without 40 μg/mL oxLDL. Scale bars, 10 μm. e Co-IP detection of the interaction between DHX9 and p65 in the nuclear fractions of macrophages by using DHX9 antibody. f P65 dimer formation was detected using naïve PAGE when macrophages were transfected with or without FLAG-DHX9 and treated with or without oxLDL (-, 0 μg/mL oxLDL; +, 40 μg/ mL oxLDL; ++, 80 μg/mL oxLDL).

Article Snippet: 20 μg of protein was loaded into a SDS gel, followed by electronic transfer onto PVDF membranes (Millipore, USA), and then incubated with primary antibodies against DHX9 (No.17721–1-AP, Proteintech), STAT1 (No.10144–2-AP, Proteintech), STAT1 phospho Ser727 (p-STAT1, No.39634, Active motif), p38 (No.14064–1-AP, Proteintech), PhosphoP38 MAPK (Thr180/Tyr182) (p-p38, No.28796–1-AP, Proteintech), JNK (No.66210–1-Ig, Proteintech), PhosphoJNK (Tyr185) (p-JNK, No.80024–1-RR, Proteintech), ERK1/2 (No.11257–1-AP, Proteintech), Phospho-ERK1/2 (Thr202/Tyr204) (p-ERK1/2, No.80031–1-RR, Proteintech), p65 (No.66535–1-Ig, Proteintech), p65 (phospho S536) (p-p65, sc-136548, Santa Cruz), GAPDH (ab8245, Abcam), flag (ab205606, Proteintech), or Lamin B1 (No.66095–1-Ig, Proteintech).

Techniques: Western Blot, Transfection, Incubation, Co-Immunoprecipitation Assay, Expressing