Journal:

Article Title: Axoplasm Isolation from Peripheral Nerve

doi: 10.1002/dneu.20755

Figure Lengend Snippet: (A) Electron micrograph of negative staining of axoplasm from the isotonic squeeze method shows a nonhomogeous preparation containing vesicular structures of different sizes. (B) Negative staining of axoplasm from the hypotonic extraction procedure shows a more homogenous preparation containing numerous small size vesicle-like structures. Scale bar in both images 200 nm. (CF) Western blot comparison of soluble and pellet fractions of axoplasms extracted by different methods and cleared by regular centrifugation or ultracentrifuge (UC). Albumin and transferrin were used to monitor serum contamination, CNPase and GFAP for Schwann cells and other glia, importins and dynein intermediate chain (IC) for cell body components and retrograde signaling complexes, and general Erk1 and Erk2 MAP kinases (gERK) as a loading control. (G) Levels of different proteins in the soluble fraction from hypotonic extraction axoplasm as a percentage of their level in isotonic squeeze axoplasm. All data was normalized to the average of the control group (isotonic squeeze axoplasm), taken as 100% in each blot. Statistics were by one-sample t-test with hypothesized population mean 100, p-value < 0.05. Note markedly reduced levels of serum and glia contaminants, versus enrichment of dynein associated proteins. 80 μg protein per lane.

Article Snippet: Mouse anti-Dynein intermediate chain clone 74.1 was from Chemicon (MAB1618), rabbit anti-NFH was from Chemicon (AB1989); mouse anti-NFH clone N52 was from Sigma; mouse anti-Importin β clone 31H4 was from Sigma (I2534); mouse anti-CNPase was from Chemicon (MAB326); mouse anti-GFAP clone G-A-5 was from Sigma (G6171); rabbit anti-albumin and rabbit anti-transferrin were from Cedarlane (CLAG5140 and GLAG5240 respectively); mouse anti-tubulin β3 was from Sigma (T2200); and rabbit anti-gERK was from Sigma (M5670).

Techniques: Negative Staining, Western Blot, Centrifugation

Journal:

Article Title: Phospholipase D2 Is Localized to the Rims of the Golgi Apparatus in Mammalian Cells

doi: 10.1091/mbc.02-04-0059

Figure Lengend Snippet: PLD2 localization with different organelles in rat NRK cells. Cells were prepared for immunofluorescence microscopy and incubated with rabbit PLD2 antibody PLD2-27 (A, D, G, and J) and costained with monoclonal antibodies to the ER marker BiP (B); the transferrin receptor (E), a marker of early endosomes and the plasma membrane; and lgp120 (H), a late endosome/lysosomal marker. (K) Cells were stained with caveolin-1, a marker for caveoli; the arrow corresponds to caveolin-1 localized to the plasma membrane. Images were merged to determine overlap between PLD2 (red) and the respective marker proteins (green; C, F, I, and L). Areas of maximal overlap are yellow. (L) Inset, enlargement of the region of overlap between PLD2 and caveolin-1 in the perinuclear Golgi apparatus. The arrow indicates areas where PLD2 puncta (red) either overlap completely with caveolin-1 (green) or are directly adjacent. Images are from projected Z-series. Bars, 10 μm.

Article Snippet: Purified monoclonal anti-rat transferrin receptor antibodies were purchased from Cedarlane Laboratories (Hornby, Ontario, Canada).

Techniques: Immunofluorescence, Microscopy, Incubation, Marker, Staining