Journal: BMC Plant Biology

Article Title: Integration of proteomic and genomic approaches to dissect seed germination vigor in Brassica napus seeds differing in oil content

doi: 10.1186/s12870-018-1624-7

Figure Lengend Snippet: The 2D-DIGE maps and the functional categorization of DEPs in germinating B. napus seeds with high and low oil content. a - e represent the 2D-DIGE maps of 12WH191 (H) and KenC8 (L) germinating seeds at 0, 12, 24, 36 and 48 HAI. f represents the functional categorization of DEPs. Arrows show the protein spots that were highly expressed in low oil-containing seeds; lines show the protein spots that were highly expressed in high oil-containing seeds

Article Snippet: For 2D-DIGE analysis, the labeled proteins were mixed with 2D-DIGE buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.4% DTT, 0.5% IPG buffer) and separated with isoelectric focusing after being loaded on an immobilized pH gradient strip (IPG, pH 4–7, 24 cm; Amersham Biosciences, Uppsala, Sweden) [ ].

Techniques: Functional Assay

Journal:

Article Title: The scavenger receptor SR-A I/II (CD204) signals via the receptor tyrosine kinase Mertk during apoptotic cell uptake by murine macrophages

doi: 10.1189/jlb.0307135

Figure Lengend Snippet: Exposure of J774 to AC induces association of SR-A and Mertk. Equal numbers of J774 (A and B) or PMø from 129P3/J or SR-A−/− mice (C) were stimulated in vitro with a fivefold excess of AC for various times as indicated, washed extensively, and then lysed in OG buffer. After preclearing, Mø lysates were immunoprecipitated (IP) using anti-Mertk (A and C) or goat anti-SR-A (B) and protein G-agarose, run on a 7.5% acrylamide gel under reducing conditions, and transferred to PVDF. Blots [immunoblots (IB)] were then blocked and probed using rat anti-SR-A (A and C) or anti-Mertk (B) and washed for 15 min using Tween-TBS. After incubation with HRP-conjugated anti-rat or anti-goat IgG in blocker, signal was detected by ECL. Finally, to demonstrate protein loading, blots were stripped and reblotted with anti-Mertk (A and C) or rat anti-SR-A (B). In each case, similar results were obtained in an additional experiment of identical design. wt, Wild-type.

Article Snippet: The following reagents were purchased from the indicated vendors: PBS without cations, DMEM, FBS, and penicillin/streptomycin from Life Technologies (Rockville, MD, USA); sodium orthovandadate, sodium fluoride, octyl-β-D-glucopyranoside (OG), fucoidan, heparin, dextran sulfate, BSA Fraction V, dexamethasone, 2-ME, glycerol, NaCl, Tris HCl, and phosphatase inhibitor cocktail II from Sigma Chemical Co. (St. Louis, MO, USA); rat anti-SR-A antibody (clone 2F8) from Serotec (Raleigh, SC, USA); goat anti-SR-A antibody and polyclonal goat anti-Mertk antibody from R&D Systems (Minneapolis, MN, USA); anti-CD36 from BD PharMingen (San Jose, CA, USA); polyclonal rabbit anti-PLCγ2 antibody (sc-407), anti-actin antibody, rat IgG, mouse IgG, protein L-agarose, protein G-agarose, and HRP-conjugated anti-rabbit and anti-goat IgG from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-phospho (p)Mertk antibody from FabGennix Inc. (Frisco, TX, USA); SDS, 0.2 μm polyvinylidene difluoride (PVDF) membrane, nonfat dry milk blocker, and 7.5% acrylamide Ready Gels from BioRad (Hercules, CA, USA); Supersignal West Femto maximum sensitivity substrate and TBS, pH 7.2, from Pierce (Rockford, IL, USA); and Kodak X-Omat AR film and eight-well Lab-Tek slides from Fisher Scientific (Chicago, IL, USA).

Techniques: In Vitro, Immunoprecipitation, Acrylamide Gel Assay, Western Blot, Incubation

Journal:

Article Title: The scavenger receptor SR-A I/II (CD204) signals via the receptor tyrosine kinase Mertk during apoptotic cell uptake by murine macrophages

doi: 10.1189/jlb.0307135

Figure Lengend Snippet: Anti-SR-A inhibits the AC-induced tyrosine phosphorylation of Mertk and PLCγ2. Equal numbers of J774 were preincubated at 37°C and 5% CO2 in medium containing 20 μg/ml rat IgG or anti-SR-A and after washing, were stimulated in vitro with a fivefold excess of AC for various times as indicated, washed extensively, and then lysed in OG buffer. After preclearing, Mø lysates were (A) immunoprecipitated using anti-pTyr antibody (anti-pY) and protein G-agarose and run on a 7.5% acrylamide gel under reducing conditions and transferred to PVDF or alternatively, (B) run directly on a 7.5% acrylamide gel. Blots were then blocked and probed using anti-Mertk (A) or anti-pMertk (B) and washed for 15 min using Tween-TBS. After incubation with HRP-conjugated anti-goat (for detection of Mertk) or anti-rabbit (for detection of PLCγ2) IgG in blocker, signal was detected by ECL. Finally, blots were stripped and reblotted with anti-PLCγ2 (A) or anti-Mertk (B). Similar results were obtained in an additional experiment of identical design.

Article Snippet: The following reagents were purchased from the indicated vendors: PBS without cations, DMEM, FBS, and penicillin/streptomycin from Life Technologies (Rockville, MD, USA); sodium orthovandadate, sodium fluoride, octyl-β-D-glucopyranoside (OG), fucoidan, heparin, dextran sulfate, BSA Fraction V, dexamethasone, 2-ME, glycerol, NaCl, Tris HCl, and phosphatase inhibitor cocktail II from Sigma Chemical Co. (St. Louis, MO, USA); rat anti-SR-A antibody (clone 2F8) from Serotec (Raleigh, SC, USA); goat anti-SR-A antibody and polyclonal goat anti-Mertk antibody from R&D Systems (Minneapolis, MN, USA); anti-CD36 from BD PharMingen (San Jose, CA, USA); polyclonal rabbit anti-PLCγ2 antibody (sc-407), anti-actin antibody, rat IgG, mouse IgG, protein L-agarose, protein G-agarose, and HRP-conjugated anti-rabbit and anti-goat IgG from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-phospho (p)Mertk antibody from FabGennix Inc. (Frisco, TX, USA); SDS, 0.2 μm polyvinylidene difluoride (PVDF) membrane, nonfat dry milk blocker, and 7.5% acrylamide Ready Gels from BioRad (Hercules, CA, USA); Supersignal West Femto maximum sensitivity substrate and TBS, pH 7.2, from Pierce (Rockford, IL, USA); and Kodak X-Omat AR film and eight-well Lab-Tek slides from Fisher Scientific (Chicago, IL, USA).

Techniques: Phospho-proteomics, In Vitro, Immunoprecipitation, Acrylamide Gel Assay, Incubation

Journal:

Article Title: The scavenger receptor SR-A I/II (CD204) signals via the receptor tyrosine kinase Mertk during apoptotic cell uptake by murine macrophages

doi: 10.1189/jlb.0307135

Figure Lengend Snippet: Mertk phosphorylation is inhibited in tissue Mø of SR-A−/− mice. (A) Coimmunoprecipitation of PMø following AC exposure. PMø from 129P3/J mice (upper gel) or SR-A−/− mice (lower gel) were exposed to fivefold excess of AC for various times as indicated. Cells were lysed, pTyr immunoprecipitates formed, and gels were probed with anti-Mertk and developed as described in the legend to Figure 3. The experiments shown were repeated in two additional experiments with similar results. (B) Western blot. PMø lysates (40 μg protein) from wild-type 129P3/J mice or SR-A−/− mice were run on a 4–20% acrylamide gel, and gels were transferred and blocked as described in the legend to Figure 4 and probed with anti-Mertk, followed by stripping and reprobing with anti-actin. (C) Western blot. PMø lysates from wild-type 129P3/J mice or SR-A−/− mice were run on a 4–20% acrylamide gel, and gels were transferred and blocked as described in the legend to Figure 4 and probed with anti-pMertk, followed by stripping and reprobing with anti-Mertk.

Article Snippet: The following reagents were purchased from the indicated vendors: PBS without cations, DMEM, FBS, and penicillin/streptomycin from Life Technologies (Rockville, MD, USA); sodium orthovandadate, sodium fluoride, octyl-β-D-glucopyranoside (OG), fucoidan, heparin, dextran sulfate, BSA Fraction V, dexamethasone, 2-ME, glycerol, NaCl, Tris HCl, and phosphatase inhibitor cocktail II from Sigma Chemical Co. (St. Louis, MO, USA); rat anti-SR-A antibody (clone 2F8) from Serotec (Raleigh, SC, USA); goat anti-SR-A antibody and polyclonal goat anti-Mertk antibody from R&D Systems (Minneapolis, MN, USA); anti-CD36 from BD PharMingen (San Jose, CA, USA); polyclonal rabbit anti-PLCγ2 antibody (sc-407), anti-actin antibody, rat IgG, mouse IgG, protein L-agarose, protein G-agarose, and HRP-conjugated anti-rabbit and anti-goat IgG from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-phospho (p)Mertk antibody from FabGennix Inc. (Frisco, TX, USA); SDS, 0.2 μm polyvinylidene difluoride (PVDF) membrane, nonfat dry milk blocker, and 7.5% acrylamide Ready Gels from BioRad (Hercules, CA, USA); Supersignal West Femto maximum sensitivity substrate and TBS, pH 7.2, from Pierce (Rockford, IL, USA); and Kodak X-Omat AR film and eight-well Lab-Tek slides from Fisher Scientific (Chicago, IL, USA).

Techniques: Phospho-proteomics, Western Blot, Acrylamide Gel Assay, Stripping Membranes