Journal:

Article Title: The nuclear factor kappa B (NF-?B): A potential therapeutic target for estrogen receptor negative breast cancers

doi: 10.1073/pnas.151257998

Figure Lengend Snippet: Inhibition of NF-κB activation and down stream events by stable expression of dominant-negative IκB-kinase β (dnIkkβ). (A Upper) dnIkkβ-Expressing transfectant cells by light microscopy (i), 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei (ii), and immunofluorescence with anti-FLAG antibody in the presence of the secondary antibody (iii). The positive signals show dnIkkβ-conjugated FLAG protein in the cytoplasm. (Lower) Processed vector-control plasmid-transfected CSMLO cells in which no FLAG protein could be detected (vi). (B Upper) Active NF-κB was determined by its [γ-32P]DNA binding activity by EMSA, in three dnIkkβ-expressing stable transfectants (dnIkkβ1-1, dnIkkβ1-3, and dnIkk-β1-5) and (Lower) in parent CSMLO cells and vector control plasmid expressing transfectant (vect1-5). (C Upper) The level of ccD1 in the same three-dnIkkβ-expressing CSMLO and vector control transfected cells, as measured by Western blot analysis. (Lower) Actin analyzed similarly by immunodetection with anti-β-actin antibody that serves as a loading control.

Article Snippet: Hybond nitrocellulose membrane and ECL (enhanced chemiluminescence) immunodetection kits were obtained from Amersham Pharmacia.

Techniques: Inhibition, Activation Assay, Expressing, Dominant Negative Mutation, Transfection, Light Microscopy, Staining, Immunofluorescence, Plasmid Preparation, Binding Assay, Activity Assay, Western Blot, Immunodetection

Journal: Journal of Biological Chemistry

Article Title: α-Synuclein in Central Nervous System and from Erythrocytes, Mammalian Cells, and Escherichia coli Exists Predominantly as Disordered Monomer

doi: 10.1074/jbc.m111.318949

Figure Lengend Snippet: FIGURE 4. Native -syn from in vivo sources co-migrates with recombinant unfolded -syn monomer. A, CN-PAGE analysis of nontransgenic and transgenic (expressing A53T human -syn) mouse brain homogenates (10 g of total protein per lane) extracted under nondenaturing conditions. 200 ng of recombinant standards were used. The blot was probed using the Syn-1 clone antibody that recognizes both human and mouse -syn. Immunodetection was performed using ECL (GE Healthcare) chemiluminescence detection reagent. Images were captured using a FujiFilm LAS-4000 luminescent image analysis system. ctx, cortex; BS, brain stem. B, same experiment was performed from rat brain homogenates obtained 4 weeks after AAV injection as described under “Experimental Procedures.” 10 g of total protein were also loaded on each lane. Western blots were processed as in A. SN, substantia nigra; ST, striatum; inj, injected. C, CN-PAGE of native -syn from human control and diseased brains, an independent mouse transgenic line, and -syn-KO (s-ko) mice (negative control). For analysis in clear native gels, 18 g of total protein (from mouse and human brain homogenates) were prepared in Novex Tris-glycine (Ntg) native sample buffer (Invitrogen) and separated by gel electrophoresis on 12% Tris-glycine gels (Invitrogen). Blots were incubated overnight at 4 °C with antibodies against -syn (Millipore). Membranes were processed using a chemiluminescence kit (Western Lightning Chemiluminescence Reagent Plus; PerkinElmer Life Sciences) and imaged using a Versadoc system (Bio-Rad). The arrow marks nonspecific bands (assigned as such because they appear in -syn-KO mouse samples). Ctl, control. D, SDS-PAGE analysis of the same samples as in C. Samples were heated for 10 min at 70 °C and then separated by gel electrophoresis on 4–12% BisTris SDS-polyacrylamide gels (Invitrogen). Western blots were also processed in the same way as in C. IB, immunoblot; N-Ac, N-acetylated -syn.

Article Snippet: Membranes were processed using a chemiluminescence kit (Western Lightning Chemiluminescence Reagent Plus; PerkinElmer Life Sciences) and imaged using a Versadoc system (Bio-Rad).

Techniques: In Vivo, Recombinant, Clear Native PAGE, Transgenic Assay, Expressing, Immunodetection, Injection, Western Blot, Control, Negative Control, Nucleic Acid Electrophoresis, Incubation, SDS Page

Journal: Journal of Biological Chemistry

Article Title: α-Synuclein in Central Nervous System and from Erythrocytes, Mammalian Cells, and Escherichia coli Exists Predominantly as Disordered Monomer

doi: 10.1074/jbc.m111.318949

Figure Lengend Snippet: FIGURE5.Recombinantandnative(endogenousandoverexpressed)-synco-migrateassinglemonomericspecies.A,SDS-PAGEofcelllysateobtained from HEK293T and HeLa cells transiently transfected or not (control) with a plasmid encoding WT human -syn. Both recombinant (N-Ac and wt) and exogenous -syn co-migrate at 15 kDa. The blot was revealed by fluorescence scanned at lower exposure, allowing the detection of exogenous -syn only. B, SDS-PAGE of five different mammalian cell lines screened for endogenous -syn expression and revealed by enhanced chemiluminescence. Native endog- enous -syn from these cells when detectable co-migrates with recombinant purified -syn at 15 kDa. C and D, same samples obtained in A and B, respectively, were resolved in 7.5% CN-PAGE and revealed as described (“Experimental Procedures”). As shown, exogenous -syn WT (C) and native -syn co-migrate as single band above 66 kDa on CN-PAGE. Endogenous -syn from the COS-7 cell line migrates slightly slower than recombinant -syn and below the recombi- nant dimers. We failed to detect -syn in CHO cell lysate in both conditions. N-Ac, recombinant purified N-acetylated -syn; Wt, control recombinant purified -syn; A140C, control recombinant purified dimers; M, -syn monomers; D, -syn dimers.

Article Snippet: Membranes were processed using a chemiluminescence kit (Western Lightning Chemiluminescence Reagent Plus; PerkinElmer Life Sciences) and imaged using a Versadoc system (Bio-Rad).

Techniques: Transfection, Control, Plasmid Preparation, Recombinant, Fluorescence, SDS Page, Expressing, Purification, Clear Native PAGE