Journal: Molecular & Cellular Proteomics

Article Title: Application of Mass Spectrometry Profiling to Establish Brusatol as an Inhibitor of Global Protein Synthesis

doi: 10.1074/mcp.m115.055509

Figure Lengend Snippet: FIG. 3. Effects of brusatol on total protein synthesis. (A) Scatterplot showing the melting temperatures of all proteins detected in both lysates and intact cells under the DMSO condition. The solid line shows the best fit with an R2 concordance of 0.43, while the red dashed line indicates a hypothetical unity response, to demonstrate that most proteins are more thermostable when heated in lysis buffer compared with intact cells. (B) The ratios of protein abundance of 3499 proteins at the 38 °C temperature comparing brusatol-treated to DMSO-treated A549 cells. Protein abundance was quantified from summed TMT reporter ion intensities of peptide-spectrum-matchings. The scatter plot shows data from two replicate measurements indicating that the majority of proteins show reduced abundance following brusatol treatment. cystatin C (red), IGFBP4 (blue), PAF (purple), and NRCAM (brown) are indicated. (C) MS abundance data for cystatin C, IGFBP4, and PAF over the temperature ranges used are shown. X axis: temperature points to heat intact cells in the CETSA assay. Y axis: protein abundance at each temperature point normalized to the average of DMSO and brusatol-treated protein abundance at 38 °C. Green: DMSO treatment; red: brusatol treatment. (D) The same lysates used for MS analysis were used for Western blotting using antibodies specific for cystatin C, IGFBP4, and PAF.

Article Snippet: Membranes were blotted with the following primary antibodies: Nrf2 (Abcam ab62352), cystatin-C (Abcam ab133495), IGFBP4 (Thermo PA5–25925), PAF1 (Bethyl A304–374A), NRCAM (ProteinTech 21608–1-AP), MCL-1 (Cell Signaling 5453), p21 (Cell Signaling 2947), Gemin-4 (Abcam ab31581), FAM96A (Acris AP51504PU-N), RL22 (Santa Cruz sc-136413), GCN1L1 (Abcam ab86139), RPL11 (Abcam ab79352), RL9 (Santa Cruz sc-292593), survivin (sc-17779), cyclin A (sc-751), and -actin (Cell Signaling 12620).

Techniques: Lysis, Quantitative Proteomics, Western Blot

Journal: Molecular & Cellular Proteomics

Article Title: Application of Mass Spectrometry Profiling to Establish Brusatol as an Inhibitor of Global Protein Synthesis

doi: 10.1074/mcp.m115.055509

Figure Lengend Snippet: FIG. 5. Brusatol-induced effects on protein abundance are independent of changes in mRNA. (A) A549 cells were treated with 0.1% DMSO, 50 nM or 500 nM brusatol or 5 or 50 g/ml cycloheximide for the indicated times before harvesting the cells. Lysates were separated by SDS-PAGE and Western blotted using the indicated antibodies. (B) mRNA levels of Nrf2, cystatin C, PAF, NRCAM, GCN1L, RL22, and RL11 were analyzed at the 30 min and 4 h time points of brusatol and cycloheximide treated cells compared with no treatment control. NTC nontargeted control siRNA. Error bars show standard deviation (n 2). (C) Protein abundance change quantified after 4-h brusatol treatment or 6-h cycloheximide treatment (data from (24)). To filter out noise and look at more profound changes, a minimum protein level change of 0.6 (2) was required for the brusatol dataset. Proteins showing greater than 3 changes were highlighted. Pearson correlation was calculated. (D) A549 cells were treated with 0.1% DMSO, 50 nM or 500 nM brusatol or 5 or 50 g/ml cycloheximide for the indicated times before harvesting the cells. Lysates were separated by SDS-PAGE and Western blotted using the indicated antibodies.

Article Snippet: Membranes were blotted with the following primary antibodies: Nrf2 (Abcam ab62352), cystatin-C (Abcam ab133495), IGFBP4 (Thermo PA5–25925), PAF1 (Bethyl A304–374A), NRCAM (ProteinTech 21608–1-AP), MCL-1 (Cell Signaling 5453), p21 (Cell Signaling 2947), Gemin-4 (Abcam ab31581), FAM96A (Acris AP51504PU-N), RL22 (Santa Cruz sc-136413), GCN1L1 (Abcam ab86139), RPL11 (Abcam ab79352), RL9 (Santa Cruz sc-292593), survivin (sc-17779), cyclin A (sc-751), and -actin (Cell Signaling 12620).

Techniques: Quantitative Proteomics, SDS Page, Western Blot, Control, Standard Deviation

Journal: Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association

Article Title: Kaempferol prevents the activation of complement C3 protein and the generation of reactive A1 astrocytes that mediate rat brain degeneration induced by 3-nitropropionic acid.

doi: 10.1016/j.fct.2022.113017

Figure Lengend Snippet: Fig. 4. Kaempferol co-administration prevents the increase in NF-κB induced by NPA treatment. (A) Representative Western blot of NF-κB and β-actin of striatum and hippocampus homogenates of rats of Control-group (CT), NPA- group (NPA), and KNPA-group (KNPA). After the acquisition of images of the Western blot with anti-NF-κB-p65 antibody, the PVDF membrane was stripped and processed for the Western blot of anti-β-actin, as indicated in the Materials and Methods. The β-actin band has been cropped from Supplementary Fig. S3 included at the end of this manuscript. The molecular weights of the protein markers (MWM) are indicated on the right-hand side. (B) A plot of the ratio of (NF-κB/β-actin) in striatum and hippocampus homogenates of rats of CT-, NPA- and KNPA-groups. The bars are the means ± SEM of the results obtained with samples of each group of rats (n = 6). (*) p < 0.05 relative to Control rats.

Article Snippet: Tissue sections were blocked with endogenous avidin/biotin blocking kit (Abcam ab 64212) and incubated with primary antibodies: dilution 1:2000 for rabbit anti-C3 antibody (Abcam ab225539, a PBSbuffered version of ab200999, containing no BSA or sodium azide), dilution 1:100 for rabbit anti-TNFα antibody (Abcam ab6671), dilution 1:50 for rabbit anti-NF-κB-p65 (Proteintech 10745-1-AP) and dilution 1:500 for rabbit anti-neurogranin (Chemicon AB5620).

Techniques: Western Blot, Control, Membrane

Journal: Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association

Article Title: Kaempferol prevents the activation of complement C3 protein and the generation of reactive A1 astrocytes that mediate rat brain degeneration induced by 3-nitropropionic acid.

doi: 10.1016/j.fct.2022.113017

Figure Lengend Snippet: Fig. 5. Representative coronal sections of the striatum (A) and hippocampus (B). Immunohistochemistry with anti-NF-κB-p65 (in red) of Control- and KNPA-groups. Double immunohistochemistry with anti-NF-κB-p65 (in red) and anti-GFAP (in blue) antibodies in the NPA group. In NPA-group, note the co-location of NF-κB-p65 and GFAP in ameboid-shape reactive A1 astrocytes (red arrowheads) in the area surrounding the core (sc) in the striatum (A) and, also, in CA1 and CA3 (Ammon’s horn) fields in the hippocampus (B). In the lesion core area (lc) of the striatum (A) no staining in cellular somas is observed with NF-κB-p65 in Control-, NPA-, and KNPA-groups. In contrast, in the hippocampus (B) pyramidal neurons (p) of CA1 and CA3 show staining with NF-κB-p65 in Control-and KNPA-groups. AL: alveus; cc: corpus callosum; CTX: Cerebral cortex; DG: dentate gyrus; lu: stratum lucidum; o: stratum oriens; ra: stratum radiatum. Red scale bars: 200 μm. Green scale bars: 10 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Tissue sections were blocked with endogenous avidin/biotin blocking kit (Abcam ab 64212) and incubated with primary antibodies: dilution 1:2000 for rabbit anti-C3 antibody (Abcam ab225539, a PBSbuffered version of ab200999, containing no BSA or sodium azide), dilution 1:100 for rabbit anti-TNFα antibody (Abcam ab6671), dilution 1:50 for rabbit anti-NF-κB-p65 (Proteintech 10745-1-AP) and dilution 1:500 for rabbit anti-neurogranin (Chemicon AB5620).

Techniques: Immunohistochemistry, Control, Staining