Journal: Human Reproduction (Oxford, England)

Article Title: Seminal plasma promotes decidualization of endometrial stromal fibroblasts in vitro from women with and without inflammatory disorders in a manner dependent on interleukin-11 signaling

doi: 10.1093/humrep/deaa015

Figure Lengend Snippet: Degradation or denaturation of seminal proteins eliminates SP-enhanced decidualization. (A) SP was mock-treated or incubated with Proteinase K (200 μg/ml) for 18 h at 37°C, followed by the addition of phenylmethylsulphonyl fluoride to inactivate the Proteinase K. Protein content was then analyzed by Coomassie staining. (B) Proteinase K treatment abrogates SP-enhanced decidualization. IGFBP1 levels were measured in supernatants of eSF cultures (Donors D and J) after 14 days of treatment with media alone, E2P4, SP, E2P4 + SP or E2P4 + Proteinase K-treated SP. *P < 0.05, **P < 0.01 and ***P < 0.001, as determined using a two-tailed Student’s t-test comparing each treatment condition (E2P4, SP, E2P4 + SP and E2P4 + Proteinase K-treated SP) to the corresponding mock-treated samples. (C) Proteinase K-treated SP is not cytotoxic to eSFs. eSF cultures set up as described in panel B were assessed for cellular viability using the CellTiter-Glo luminescent assay. (D) Denaturation of seminal proteins by boiling diminishes SP-enhanced decidualization. IGFBP1 levels were measured in supernatants of eSF cultures (Donors C and F) after 14 days of treatment with media alone, E2P4, SP, E2P4 + SP or E2P4 + boiled SP. **P < 0.01, ***P < 0.001 and ****P < 0.0001 as determined using a two-tailed Student’s t-test comparing each treatment condition (E2P4, SP, E2P4 + SP and E2P4 + boiled SP) to the corresponding mock-treated samples. (E) Boiled SP is not cytotoxic to eSFs. eSF cultures set up as described in panel D were assessed for cellular viability using the CellTiter-Glo luminescent assay.

Article Snippet: SP protein degradation For assessing the role of SP proteins in SP-induced changes in eSFs, SP proteins were denatured by heating at 100°C for 1 h or degraded by treatment with Proteinase K (200 μg/ml) (Sigma-Aldrich) for 18 h at 37°C followed by the addition of phenylmethylsulphonyl fluoride at a final concentration of 5 mM (Roche, Basel, Switzerland) to inactivate the Proteinase K. Protein degradation by Proteinase K was confirmed by running samples at 100 V for 1 h on a Bis-Tris 4–12% gradient protein gel (ThermoFisher) followed by staining with Bio-SafeTM Coomassie (Bio-Rad, Hercules, CA, USA) following the manufacturer’s protocol.

Techniques: Incubation, Staining, Two Tailed Test, Luminescence Assay

Journal:

Article Title: Flagellin Diversity in Clostridium botulinum Groups I and II: a New Strategy for Strain Identification ▿

doi: 10.1128/AEM.02623-06

Figure Lengend Snippet: SDS-PAGE profiles of flagellar proteins. (A) Group I flagellins. Lane 1, Hall A; lane 2, FE9504ACG; lane 3, 17A; lane 4, FE9909ACS Alberta; lane 5, FE0303A1YO; lane 6, MUL0109ASA; lane 7, PC0101AJO; lane 8, MRB; lane 9, FE0507BLP; lane 10, PA9508B; lane 11, 920A276; lane 12, FE9904BMT; lane 13, FE9508BPD; and lane 14, Langeland. (B) Group II flagellins. Lane 1, 17B; lane 2, KapB3; lane 3, ERuss; lane 4, Bennett; lane 5, GA9709EHS; and lane 6, 610F. Molecular masses in kilodaltons are indicated by arrows. Serotypes of strains are indicated by a letter below each lane. All predominant bands were identified as flagellin by mass spectrometry, with the exception of the 30-kDa molecular mass band in panel B, lanes 1, 2, and 6 (see Table ​Table22 and the text). The minor band at 38 kDa in lanes 1, 2, and 6 was also identified as flagellin.

Article Snippet: Protein bands excised from Bio-Safe Coomassie blue (Bio-Rad)-stained SDS-PAGE gels were cut into 1-mm cubes.

Techniques: SDS Page, Mass Spectrometry

Journal: Bio-protocol

Article Title: In Gel Kinase Assay

doi: 10.21769/BioProtoc.2170

Figure Lengend Snippet: Wild-type (Col-0) and mutant lines: pyr/pyl112458 and snrk2.2/2.3/2.6 were mock or ABA (100 µM) treated for 30 min. A. SnRK2.2/2.3/2.6 kinase activity is detectable in ABA-treated (100 µM ABA) Col-0 and it is not detected in Col-0 control conditions (mock treated) neither ABA-treated (100 µM ABA) mutant line snrk2.2/2.3/2.6 nor sextuple mutant line pyr/pyl112458. Recombinant OST1/SnRK2.6-6HIS was used as a positive control. B. A parallel Coomassie stained SDS-PAGE. MW: molecular weight [kD]: 72, 55 and 43. RuBP-L: RuBisCO large chain.

Article Snippet: Eppendorf and Falcon tubes (1.5 ml and 50 ml respectively; generic) Pipettes and tips (generic) Micropore tape (3M, catalog number: 1530-0) Sterile round Petri dishes (100 mm diameter x 20 mm height; generic: e.g. , Greiner Bio One, catalog number: 664161) Extra thick blot filter paper, 7.5 x 10 cm (Bio-Rad Laboratories, catalog number: 1703965) Arabidopsis thaliana seeds: Col-0 (NASC, N1093), pyr/pyl112458 and snrk2.2/2.3/2.6 (Gonzalez-Guzman et al. , 2012) Optional: purified recombinant kinase OST1 (SnRK2.6-6his; homemade: Vlad et al. , 2009 and 2012) Sterile deionized water (generic) Liquid N 2 (generic) Ice (generic) ABA (+cis, trans-Abscisic acid) (BIOSYNTH, catalog number: A-0120) Bio-Rad Protein Assay Kit I (Bio-Rad Laboratories, catalog number: 5000001) Bio-Safe TM Coomassie Stain reagent (Bio-Rad Laboratories, catalog number: 1610786) ∆C-ABF2 peptide (amino acids 1 to 173; 18.4 KD; homemade: Dupeux et al. , 2011 ; Antoni et al. , 2012 ) Note: Commercially available alternative MBP (myelin basic protein) (Sigma-Aldrich, catalog number: M1891).

Techniques: Mutagenesis, Activity Assay, Control, Recombinant, Positive Control, Staining, SDS Page, Molecular Weight