Journal: PLoS ONE

Article Title: Comparative Expression Profiling of Distinct T Cell Subsets Undergoing Oxidative Stress

doi: 10.1371/journal.pone.0041345

Figure Lengend Snippet: Representative protein expression profiles of CD45RA + T cells post separation by two-dimensional gel electrophoresis in 16% T, 2.5% C polyacrylamide gels, The staining procedures (A) silver staining, (B) DIGE minimal staining and (C) DIGE saturating staining were performed as outlined in the Materials an methods section. The gels were loaded with 150 µg, 20 µg or 1 µg of total lysate, respectively. The numbers boxed in white squares indicate the relative localization for the panel of selected differentially expressed targets (1 – GSTO1, 2 – LIMS1, 3 – SODM, 4 – PROF1, 5 – PRDX2, 6 – GDIR1, 7 – GDIR2 and 8 – THIO). The pH gradient used in the first dimension (x axis) as well as the range of the size fractionation achieved in the second dimension (y axis) are indicated.

Article Snippet: The membranes were subsequently serially incubated with target-specific antibodies directed against thioredoxin (THIO) (ab16835, Abcam plc, Cambridge, UK; dilution 1∶2000), glutathione S-transferase O1 (GSTO1) (LS-C15465, Life Span Biosciences Inc, Seattle, WA, USA; dilution 1∶2000), peroxiredoxin 2 (PRDX2) (ab15572, Abcam; dilution 1∶10000), profilin (PROF1) (sc-30521, Santa Cruz Biotechnology Inc, Heidelberg, Germany; dilution 1∶500), LIMS1 (Pinch 1) (sc-47912, Santa Cruz), Rho GDP-dissociation inhibitor 1 (GDIR1) (10509-1-Ig, Proteintech Europe Ltd, Manchester, UK; dilution 1∶500), Rho GDP-dissociation inhibitor 2 (GDIR2) (ab15198, Abcam; dilution 1∶200) or superoxide dismutase 2 (SODM) (LF-MA0030, Lab Frontier Life Science, Seoul, Korea; dilution 1∶2000).

Techniques: Expressing, Two-Dimensional Gel Electrophoresis, Electrophoresis, Staining, Silver Staining, Fractionation

Journal: PLoS ONE

Article Title: Comparative Expression Profiling of Distinct T Cell Subsets Undergoing Oxidative Stress

doi: 10.1371/journal.pone.0041345

Figure Lengend Snippet: Functional classification of differentially expressed proteins.

Article Snippet: The membranes were subsequently serially incubated with target-specific antibodies directed against thioredoxin (THIO) (ab16835, Abcam plc, Cambridge, UK; dilution 1∶2000), glutathione S-transferase O1 (GSTO1) (LS-C15465, Life Span Biosciences Inc, Seattle, WA, USA; dilution 1∶2000), peroxiredoxin 2 (PRDX2) (ab15572, Abcam; dilution 1∶10000), profilin (PROF1) (sc-30521, Santa Cruz Biotechnology Inc, Heidelberg, Germany; dilution 1∶500), LIMS1 (Pinch 1) (sc-47912, Santa Cruz), Rho GDP-dissociation inhibitor 1 (GDIR1) (10509-1-Ig, Proteintech Europe Ltd, Manchester, UK; dilution 1∶500), Rho GDP-dissociation inhibitor 2 (GDIR2) (ab15198, Abcam; dilution 1∶200) or superoxide dismutase 2 (SODM) (LF-MA0030, Lab Frontier Life Science, Seoul, Korea; dilution 1∶2000).

Techniques: Functional Assay

Journal: PLoS ONE

Article Title: Comparative Expression Profiling of Distinct T Cell Subsets Undergoing Oxidative Stress

doi: 10.1371/journal.pone.0041345

Figure Lengend Snippet: Relevant characteristics and MALDI peptide fingerprinting data for the subset of verified proteins.

Article Snippet: The membranes were subsequently serially incubated with target-specific antibodies directed against thioredoxin (THIO) (ab16835, Abcam plc, Cambridge, UK; dilution 1∶2000), glutathione S-transferase O1 (GSTO1) (LS-C15465, Life Span Biosciences Inc, Seattle, WA, USA; dilution 1∶2000), peroxiredoxin 2 (PRDX2) (ab15572, Abcam; dilution 1∶10000), profilin (PROF1) (sc-30521, Santa Cruz Biotechnology Inc, Heidelberg, Germany; dilution 1∶500), LIMS1 (Pinch 1) (sc-47912, Santa Cruz), Rho GDP-dissociation inhibitor 1 (GDIR1) (10509-1-Ig, Proteintech Europe Ltd, Manchester, UK; dilution 1∶500), Rho GDP-dissociation inhibitor 2 (GDIR2) (ab15198, Abcam; dilution 1∶200) or superoxide dismutase 2 (SODM) (LF-MA0030, Lab Frontier Life Science, Seoul, Korea; dilution 1∶2000).

Techniques:

Journal: PLoS ONE

Article Title: Comparative Expression Profiling of Distinct T Cell Subsets Undergoing Oxidative Stress

doi: 10.1371/journal.pone.0041345

Figure Lengend Snippet: Combined expression pattern analysis for the panel of validated targets in independent 2-DE analyses.

Article Snippet: The membranes were subsequently serially incubated with target-specific antibodies directed against thioredoxin (THIO) (ab16835, Abcam plc, Cambridge, UK; dilution 1∶2000), glutathione S-transferase O1 (GSTO1) (LS-C15465, Life Span Biosciences Inc, Seattle, WA, USA; dilution 1∶2000), peroxiredoxin 2 (PRDX2) (ab15572, Abcam; dilution 1∶10000), profilin (PROF1) (sc-30521, Santa Cruz Biotechnology Inc, Heidelberg, Germany; dilution 1∶500), LIMS1 (Pinch 1) (sc-47912, Santa Cruz), Rho GDP-dissociation inhibitor 1 (GDIR1) (10509-1-Ig, Proteintech Europe Ltd, Manchester, UK; dilution 1∶500), Rho GDP-dissociation inhibitor 2 (GDIR2) (ab15198, Abcam; dilution 1∶200) or superoxide dismutase 2 (SODM) (LF-MA0030, Lab Frontier Life Science, Seoul, Korea; dilution 1∶2000).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Comparative Expression Profiling of Distinct T Cell Subsets Undergoing Oxidative Stress

doi: 10.1371/journal.pone.0041345

Figure Lengend Snippet: Combined expression pattern analysis for the panel of validated targets in independent Western blot analyses.

Article Snippet: The membranes were subsequently serially incubated with target-specific antibodies directed against thioredoxin (THIO) (ab16835, Abcam plc, Cambridge, UK; dilution 1∶2000), glutathione S-transferase O1 (GSTO1) (LS-C15465, Life Span Biosciences Inc, Seattle, WA, USA; dilution 1∶2000), peroxiredoxin 2 (PRDX2) (ab15572, Abcam; dilution 1∶10000), profilin (PROF1) (sc-30521, Santa Cruz Biotechnology Inc, Heidelberg, Germany; dilution 1∶500), LIMS1 (Pinch 1) (sc-47912, Santa Cruz), Rho GDP-dissociation inhibitor 1 (GDIR1) (10509-1-Ig, Proteintech Europe Ltd, Manchester, UK; dilution 1∶500), Rho GDP-dissociation inhibitor 2 (GDIR2) (ab15198, Abcam; dilution 1∶200) or superoxide dismutase 2 (SODM) (LF-MA0030, Lab Frontier Life Science, Seoul, Korea; dilution 1∶2000).

Techniques: Expressing, Western Blot

Journal: PLoS ONE

Article Title: Comparative Expression Profiling of Distinct T Cell Subsets Undergoing Oxidative Stress

doi: 10.1371/journal.pone.0041345

Figure Lengend Snippet: Western blot analyses were performed by serially probing sets of 3 membranes per donor as outlined in the Material and methods section. The expression levels of GDIR1, GSTO1, LIMS1 and PROF1 were analyzed blot 1 (upper panel), GDIR2, PRDX2 and THIO on blot 2 (middle panel) and SODM on blot 3 (lower level) of the respective membrane set. The relative expression levels were defined by densitometric quantification normalized to the corresponding beta-actin signals, co-detected on each membrane. Aliquots of 15 µg total lysate per sample/lane were loaded onto Tris-Tricine gels (16%T/2.5%C) and subsequently separated and processed as outlined in the Materials and method section. Lysates generated form untreated CD45RA + and CD45RO + T cells were throughout the set of membranes loaded onto the lanes 1 and 3 respectively, whereas lysates generated from the corresponding T cell subsets exposure for 3 h to 5 µM H 2 O 2 were loaded onto the lanes 2 and 4 as indicated. The protein entry names of the targets (left) and their corresponding molecular weight (right) are listed.

Article Snippet: The membranes were subsequently serially incubated with target-specific antibodies directed against thioredoxin (THIO) (ab16835, Abcam plc, Cambridge, UK; dilution 1∶2000), glutathione S-transferase O1 (GSTO1) (LS-C15465, Life Span Biosciences Inc, Seattle, WA, USA; dilution 1∶2000), peroxiredoxin 2 (PRDX2) (ab15572, Abcam; dilution 1∶10000), profilin (PROF1) (sc-30521, Santa Cruz Biotechnology Inc, Heidelberg, Germany; dilution 1∶500), LIMS1 (Pinch 1) (sc-47912, Santa Cruz), Rho GDP-dissociation inhibitor 1 (GDIR1) (10509-1-Ig, Proteintech Europe Ltd, Manchester, UK; dilution 1∶500), Rho GDP-dissociation inhibitor 2 (GDIR2) (ab15198, Abcam; dilution 1∶200) or superoxide dismutase 2 (SODM) (LF-MA0030, Lab Frontier Life Science, Seoul, Korea; dilution 1∶2000).

Techniques: Western Blot, Expressing, Generated, Molecular Weight

Journal: PLoS ONE

Article Title: Proteomic Analysis of Adrenocorticotropic Hormone Treatment of an Infantile Spasm Model Induced by N -Methyl-d-Aspartic Acid and Prenatal Stress

doi: 10.1371/journal.pone.0045347

Figure Lengend Snippet: (a) The proteins were analyzed using 12% (SDS–PAGE) and western blot analysis. GAPDH was used for normalization. (b) The intensities of ANXA3, ARHGDIA, and CFL1 expression from the immunoblots are shown in (a), as quantified using densitometric analysis. (c) The spots of ANXA3, ARHGDIA, and CFL1 are marked by arrows on the 2DE gel.

Article Snippet: Airy, MD), anti-ARHGDIA (Proteintech, Chicago, IL), and anti-CFL1 (Bioworld) antibodies (1∶500 in TBST) for 1.5 h at room temperature.

Techniques: SDS Page, Western Blot, Expressing