Journal: Molecular and Cellular Endocrinology

Article Title: Exposure to chemical cocktails before or after conception – The effect of timing on ovarian development ☆

doi: 10.1016/j.mce.2013.06.016

Figure Lengend Snippet: Fetal ovarian proteins exhibiting differential expression following continuous or cross-over exposure to chemical cocktails in sewage sludge fertiliser. The accession number is derived from NCBI. Fold change relative to the normalised spot volumes for the CC group are increased if marked “+” and decreased if marked “−“. Fold-change values attaining statistical significance are highlighted in bold. P values are derived by post hoc tests of log-normalised spot volumes. Spots containing significant protein matches that cannot be discriminated between are marked with * next to the spot number. Where the same protein is identified in different spots vertical lines join the spot numbers.

Article Snippet: Membranes were blocked (1 h, room temperature) with Odyssey Blocking Buffer, (927–4000: LICOR + PBS) before incubation with the following mouse monoclonal primary antibodies at 4 °C overnight: (i) Heat shock protein 60 (HSP60), 1:1,000 (ab1819, Abcam Cambridgeshire, UK); (ii) HSP70, 1:1,000 (ab47455, Abcam); (iii) HSP90, 1:1,000 (H1775, Sigma–Aldrich,Dorset, UK); (iv) Major Vault Protein (MVP), 1 μg/ml (ab14562, Abcam); Gelsolin (GSN) 3 μg/ml (ab55070, Abcam); (v) HSP90, 1:1,000 (Sigma–Aldrich, UK) (vi) and goat HSPA4L 1 μg/ml, (LS-B2621, Life Span Biosciences Inc, Seattle,USA); (vii) PTEN 2 μg/ml (ab24367, Abcam).

Techniques: Quantitative Proteomics, Derivative Assay, Activity Assay, Binding Assay, Membrane, Transduction, Conjugation Assay

Journal: Molecular and Cellular Endocrinology

Article Title: Exposure to chemical cocktails before or after conception – The effect of timing on ovarian development ☆

doi: 10.1016/j.mce.2013.06.016

Figure Lengend Snippet: Sewage sludge exposure affects MVP, HSP90 and HSP70 proteins in fetal ovaries, quantified by Western blot. (A) Representative bands for each treatment group for each Western blot, including β-actin load control. These bands are all from the same 4 ovaries and all available ovaries were used for these Western blots. (B–F) Quantitation of 5 ovarian proteins shown as box and whisker plots. The band volume for each protein was normalised against β-actin for the same lane (i.e. same ovary) and then expressed relative to the mean normalised band volume of the CC treatment group. The horizontal line in the boxes show the median values, with the limits of the boxes showing the 25% and 75% quantiles and the whiskers showing the 10% and 90% quantiles. Common superscripts between groups, for each protein, denote statistically significant differences at P < 0.05. Where there are no superscripts p values are >0.05. (G) Quantification of MVP immunopositive and immunonegative oocytes shows that there were no significant exposure effects on the density of MVP immunopositive oocytes or the ratio between immunonegative and immunopositive oocytes, although the latter tended to be higher in the CT and TC groups.

Article Snippet: Membranes were blocked (1 h, room temperature) with Odyssey Blocking Buffer, (927–4000: LICOR + PBS) before incubation with the following mouse monoclonal primary antibodies at 4 °C overnight: (i) Heat shock protein 60 (HSP60), 1:1,000 (ab1819, Abcam Cambridgeshire, UK); (ii) HSP70, 1:1,000 (ab47455, Abcam); (iii) HSP90, 1:1,000 (H1775, Sigma–Aldrich,Dorset, UK); (iv) Major Vault Protein (MVP), 1 μg/ml (ab14562, Abcam); Gelsolin (GSN) 3 μg/ml (ab55070, Abcam); (v) HSP90, 1:1,000 (Sigma–Aldrich, UK) (vi) and goat HSPA4L 1 μg/ml, (LS-B2621, Life Span Biosciences Inc, Seattle,USA); (vii) PTEN 2 μg/ml (ab24367, Abcam).

Techniques: Western Blot, Control, Quantitation Assay, Whisker Assay

Journal: Molecular and Cellular Endocrinology

Article Title: Exposure to chemical cocktails before or after conception – The effect of timing on ovarian development ☆

doi: 10.1016/j.mce.2013.06.016

Figure Lengend Snippet: Networks affected by sewage sludge exposure. Genes shown in bold are those, or their products, identified as significantly altered in at least one treatment group compared with controls (CC) in the present study. Analysis was performed using IPA.

Article Snippet: Membranes were blocked (1 h, room temperature) with Odyssey Blocking Buffer, (927–4000: LICOR + PBS) before incubation with the following mouse monoclonal primary antibodies at 4 °C overnight: (i) Heat shock protein 60 (HSP60), 1:1,000 (ab1819, Abcam Cambridgeshire, UK); (ii) HSP70, 1:1,000 (ab47455, Abcam); (iii) HSP90, 1:1,000 (H1775, Sigma–Aldrich,Dorset, UK); (iv) Major Vault Protein (MVP), 1 μg/ml (ab14562, Abcam); Gelsolin (GSN) 3 μg/ml (ab55070, Abcam); (v) HSP90, 1:1,000 (Sigma–Aldrich, UK) (vi) and goat HSPA4L 1 μg/ml, (LS-B2621, Life Span Biosciences Inc, Seattle,USA); (vii) PTEN 2 μg/ml (ab24367, Abcam).

Techniques: Ubiquitin Proteomics, Modification

Journal: Toxicology

Article Title: Gender-Specific Expression of ATP-binding Cassette ( Abc ) Transporters and Cytoprotective Genes in Mouse Choroid Plexus

doi: 10.1016/j.tox.2017.05.019

Figure Lengend Snippet: Description of primary and secondary antibodies for immunoblots

Article Snippet: Tm/T Primary and secondary blocking buffer solution Mrp1 ab24102 1:500 Abcam 190 Anti-rat S 1: 2000 RT/ 1hr 5 % NFDM Mrp2 ab3373 1:500 4°C/ ON Abcam 190 Anti-mouse S 1:2000 RT/ 1hr 5 % NFDM Mrp4 ab15602 1:500 4°C/ ON Abcam 160 Anti-rat S 1:2000 RT/ 1hr 5 % NFDM Mrp5 ab24107 1:500 4°C/ ON Abcam 160 Anti-rat S 1:2000 RT/ 1hr 5 % NFDM Nrf2 8882S 1:500 4°C/ ON Cell Signaling ~100 Anti-rat S 1:2000 RT/ 1hr 5 % BSA Gclc - 1:10000 4°C/ ON Washington ~75 Anti-rabbit S 1:20000 RT/ 1hr 5 % NFDM Ho-1 SPA-895 1:5000 4°C/ ON Stressgen Bioreagents ~33 Anti-rabbit 1:2000 RT/ 1hr 5 % NFDM Nqo1 ab2346 1:1000 4°C/ ON Abcam ~32 Anti-goat S 1:10000 RT/ 1hr 5 % NFDM β-actin ab8227 1:3500 4°C/ 1hr Abcam 42 Anti-rabbit S 1:2000 RT/ 1hr 5 % NFDM Open in a separate window NFDM, non-fat dry milk BSA, bovine serum albumin S Antibody purchase from Sigma Con, Antibody concentration Tm, incubation temperature T, incubation time ON, overnight RT, room temperature Description of primary and secondary antibodies for immunoblots

Techniques: Molecular Weight, Blocking Assay

Journal: Toxicology

Article Title: Gender-Specific Expression of ATP-binding Cassette ( Abc ) Transporters and Cytoprotective Genes in Mouse Choroid Plexus

doi: 10.1016/j.tox.2017.05.019

Figure Lengend Snippet: Primer sequences for quantitative real-time-PCR

Article Snippet: Tm/T Primary and secondary blocking buffer solution Mrp1 ab24102 1:500 Abcam 190 Anti-rat S 1: 2000 RT/ 1hr 5 % NFDM Mrp2 ab3373 1:500 4°C/ ON Abcam 190 Anti-mouse S 1:2000 RT/ 1hr 5 % NFDM Mrp4 ab15602 1:500 4°C/ ON Abcam 160 Anti-rat S 1:2000 RT/ 1hr 5 % NFDM Mrp5 ab24107 1:500 4°C/ ON Abcam 160 Anti-rat S 1:2000 RT/ 1hr 5 % NFDM Nrf2 8882S 1:500 4°C/ ON Cell Signaling ~100 Anti-rat S 1:2000 RT/ 1hr 5 % BSA Gclc - 1:10000 4°C/ ON Washington ~75 Anti-rabbit S 1:20000 RT/ 1hr 5 % NFDM Ho-1 SPA-895 1:5000 4°C/ ON Stressgen Bioreagents ~33 Anti-rabbit 1:2000 RT/ 1hr 5 % NFDM Nqo1 ab2346 1:1000 4°C/ ON Abcam ~32 Anti-goat S 1:10000 RT/ 1hr 5 % NFDM β-actin ab8227 1:3500 4°C/ 1hr Abcam 42 Anti-rabbit S 1:2000 RT/ 1hr 5 % NFDM Open in a separate window NFDM, non-fat dry milk BSA, bovine serum albumin S Antibody purchase from Sigma Con, Antibody concentration Tm, incubation temperature T, incubation time ON, overnight RT, room temperature Description of primary and secondary antibodies for immunoblots

Techniques:

Journal: Toxicology

Article Title: Gender-Specific Expression of ATP-binding Cassette ( Abc ) Transporters and Cytoprotective Genes in Mouse Choroid Plexus

doi: 10.1016/j.tox.2017.05.019

Figure Lengend Snippet: Gene or protein expression was normalized to housekeeping gene β-actin. (A) Relative mRNA expression of Nrf2, Ho-1, Nqo1 and Gclc. (B) Individual blots with corresponding β-actin together with a summary of relative protein expressions. The data are presented as means ± SE (n=4, qPCR; n =8, immunoblots). Asterisks (*) represent a statistical difference between female and male (p ≤ 0.05).

Article Snippet: Tm/T Primary and secondary blocking buffer solution Mrp1 ab24102 1:500 Abcam 190 Anti-rat S 1: 2000 RT/ 1hr 5 % NFDM Mrp2 ab3373 1:500 4°C/ ON Abcam 190 Anti-mouse S 1:2000 RT/ 1hr 5 % NFDM Mrp4 ab15602 1:500 4°C/ ON Abcam 160 Anti-rat S 1:2000 RT/ 1hr 5 % NFDM Mrp5 ab24107 1:500 4°C/ ON Abcam 160 Anti-rat S 1:2000 RT/ 1hr 5 % NFDM Nrf2 8882S 1:500 4°C/ ON Cell Signaling ~100 Anti-rat S 1:2000 RT/ 1hr 5 % BSA Gclc - 1:10000 4°C/ ON Washington ~75 Anti-rabbit S 1:20000 RT/ 1hr 5 % NFDM Ho-1 SPA-895 1:5000 4°C/ ON Stressgen Bioreagents ~33 Anti-rabbit 1:2000 RT/ 1hr 5 % NFDM Nqo1 ab2346 1:1000 4°C/ ON Abcam ~32 Anti-goat S 1:10000 RT/ 1hr 5 % NFDM β-actin ab8227 1:3500 4°C/ 1hr Abcam 42 Anti-rabbit S 1:2000 RT/ 1hr 5 % NFDM Open in a separate window NFDM, non-fat dry milk BSA, bovine serum albumin S Antibody purchase from Sigma Con, Antibody concentration Tm, incubation temperature T, incubation time ON, overnight RT, room temperature Description of primary and secondary antibodies for immunoblots

Techniques: Expressing, Western Blot