Journal: Molecular Biology of the Cell
Article Title: The endoplasmic reticulum and casein-containing vesicles contribute to milk fat globule membrane
Figure Lengend Snippet: Isolation and quality of the purified CLDs from mouse lactating mammary gland. (A) Mammary acini purified from mouse mammary gland at day 10 of lactation were washed and homogenized. Total lysate (T) was centrifuged at 800 × g for 10 min at 4°C. The postnuclear supernatant (S1) was subsequently centrifuged at 110,000 × g for 1 h at 4°C to isolate cellular membrane (P2) and soluble material (S2). After centrifugation of the total lysate at 274,000 × g for 1 h at 4°C, the top white layer was the CLD fraction. (B) Isolated CLDs were analyzed by differential interference contrast microscopy (a; nt, not treated) and fluorescence microscopy after BODIPY 493/503 staining (lipids; b, e, h, k). CLDs were counterstained with Alexa Fluor 594–conjugated WGA (d), rhodamine-conjugated phalloidin (actin; g), or Alexa 594–conjugated CTxB (GM1; j) and merged (c, f, i, l) in order to visualize potential contaminations. Scale bar, 10 μm. (C) Proteins extracted from the different fractions were separated by SDS–PAGE and stained with Coomassie blue. Note the distinct banding pattern of CLDs. (D) The same protein samples were also subjected to Western blotting to test for contamination from other cellular fractions. Specific antibodies were used to probe for marker proteins of different cellular organelles/fractions: PLIN2 (CLD protein), BTN1 (MFG protein), E-cadherin (PM), β-actin (cytosol), PdiA3 and GRP78 (ER lumen), calnexin and Stx-18 (ER membrane), and GM130 (Golgi). Note the strong enrichment of PLIN2 in the CLD fraction. BTN1, butyrophilin; E-Cad, E-cadherin; GM130, Golgi matrix protein 130; GRP78, glucose-regulated protein 78; M, whole milk; P1, pellet 1; P2, pellet 2; PdiA3, protein disulfide isomerase A3; PLIN2, perilipin2; S1, supernatant 1; S2, supernatant 2; Stx-18, syntaxin 18; T, total extract.
Article Snippet: Western blot A 20-μg amount of total protein from the MFGM fractions was analyzed by SDS-polyacrylamide 12% gel electrophoresis (SDS–PAGE) and transferred onto Hybond nitrocellulose membrane (Amersham).
Techniques: Isolation, Purification, Centrifugation, Microscopy, Fluorescence, Staining, Whole Genome Amplification, SDS Page, Western Blot, Marker