Journal: Molecular Reproduction and Development

Article Title: The seminal acrosin‐inhibitor ClTI1/SPINK2 is a fertility‐associated marker in the chicken

doi: 10.1002/mrd.23153

Figure Lengend Snippet: Effect of SPINK2 and α1‐antitrypsin on serine protease activity in sperm extracts. Twenty micrograms of proteins extracted from sperm were incubated with increasing amounts (0–800 nM) of purified SPINK2 (■) and α1‐antitrypsin (○) in a 50 mM Tris‐HCl buffer, pH 8.0 at 37°C for 20 min. The percentage of remaining sperm protease activity is indicated in y ‐axis and inhibitor concentration (nM) is indicated in x ‐axis

Article Snippet: For inhibition assays, 20 µg of proteins extracted from sperm or 200 ng of proteins from affinity chromatography fraction were incubated with increasing amounts (0–800 nM) of purified SPINK2 or α1‐antitrypsin (MP Biomedicals, Illkirch, France) in a 50 mM Tris‐HCl buffer, pH 8.0 at 37°C for 20 min.

Techniques: Activity Assay, Incubation, Purification, Concentration Assay

Journal:

Article Title: Characterization of an ERAD pathway for non-glycosylated BiP substrates which requires Herp

doi: 10.1016/j.molcel.2007.09.012

Figure Lengend Snippet: NS1 κ LC expressed in P3U.1 cells (A), RE61 λ LC and BiP (C) or HA-γ V-CH1 (E) were transiently co-expressed with or without Herp-FLAG in 293T cells. At 24 hr post-transfection, cell extracts were prepared from cells treated with or without lactacystin and immunoprecipitated with anti-κ LC antiserum (A), anti-λ LC antiserum (C) or anti-HA antibody (E). Cell extracts and precipitated samples were subjected to immunoblot analyses as indicated. Non-secreted λ LC RE61 (B) or HA-γ V-CH1 (D) were transiently expressed in 293T cells. At 24 hr post-transfection, cells were labeled with 35S-methionine/cysteine for 15 min and chased for the indicated times in the presence or absence of lactacystin. Immunoprecipitated samples from cell extracts were subjected to SDS-PAGE, followed by autoradiography. The signals for λ LC RE61 (B) or HA-γ V-CH1 (D) were quantified as expressed as a percent of that present at t=0. The values are shown at the bottom of each lane. The α1-antitrypsin (AAT) NHK variant (F) or Z variant (G) was transiently co-expressed with or without Herp-FLAG in 293T cells. At 24 hr post-transfection, cell extracts were prepared after treatment with or without tunicamycin for 3 hr and subjected to immunoprecipitation with anti-α1-antitrypsin antiserum. Cell extracts and precipitated samples were subjected to immunoblot analysis as indicated.

Article Snippet: All other antibodies were purchased from companies; anti-mouse IgG (Igγ and κ) and anti-mouse IgM (Igμ and λ) (Southern Biotech), anti-actin, anti-Hsc70, and anti-FLAG D-8 (Santa Cruz), anti-ubiquitinated proteins FK2, anti-HC8, and anti-S1 (BIOMOL), and anti-α1 antitrypsin (MP Biomedicals).

Techniques: Transfection, Immunoprecipitation, Western Blot, Labeling, SDS Page, Autoradiography, Variant Assay