Journal: Nature Communications

Article Title: PRL3-zumab as an immunotherapy to inhibit tumors expressing PRL3 oncoprotein

doi: 10.1038/s41467-019-10127-x

Figure Lengend Snippet: Intracellular PRL3 is externalized for PRL3-zumab binding. a Methodology for cell surface analysis of MHCC-LM3 cultured cells (CC) and tumor cells. b – d “Surface” detection of nonspecific control antigens ( b ), EGFR ( c ), or PRL3 ( d ) were detected by fluorescence-activated cell sorting (FACS) analysis using polyclonal human hIgG, anti-EGFR antibody (cetuximab), or anti-PRL3 antibody (PRL3-zumab), respectively. Representative FACS profiles from four biological replicates are shown. e Mean percentage ± s.d. of surface positive (surface+) live cells for each antigen were calculated by dividing the surface antigen-positive live cells (upper left quadrant) by total live cells (sum of both upper and lower left quadrants) in b – d . f Background-corrected values from e were normalized to CC surface expression levels for EGFR (filled circles) and PRL3 (filled squares). The mean fold change was calculated by the Student’s t test (mean ± s.d., n = 4 biologically independent samples). P values as indicated for each antigen. g Background-corrected values of MHCC-LM3 cells cultured under “Normal” vs. “Serum-starved” conditions for 72 h were normalized to “Normal” surface+ cell percentages for each antigen. The mean fold-change was calculated by the Student’s t test (mean ± s.d.) for EGFR (filled circles; n = 3 independent samples) and PRL3 (filled squares; n = 4 independent samples). P values as indicated for each antigen. Source data are provided as a Source Data file

Article Snippet: Anti-CD16/CD32 antibody (clone 2.4G2), polyclonal human IgG, and polyclonal mouse IgG were purchased from Bio X Cell.

Techniques: Binding Assay, Cell Culture, Control, Fluorescence, FACS, Expressing

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

Journal:

Article Title: Expansion of a CD28-Intermediate Subset among CD8 T Cells in Patients with Infectious Mononucleosis

doi: 10.1128/JVI.76.13.6602-6608.2002

Figure Lengend Snippet: The CD28-int CD8 T cells have cytotoxic activity specific for autologous EBV-transformed B cells. (A) Separation of CD8 T cells from a patient with AIM into CD28-positive, -int, and -negative fractions. Flow cytometric analyses of CD8 and CD28 expressions showed that CD8 T cells could be fractionated into CD28-positive (a), -int (b), and -negative (c) CD8 subsets. Throughout the culture period the cells were incubated in the presence of IL-2 (100 U/ml) and IL-10 (100 U/ml) to prevent apoptosis (23). Typical results of three different AIM patients are shown. (B) Comparison of cytotoxic activities among CD8/CD28 subsets. Each CD8/CD28 subset was separately isolated by two cycles of electronic sorting. The three fractionated CD8 subsets (a, b, and c) were tested in triplicate for cytotoxicity against 51Cr-labeled autologous BLC with various effector/target ratios (1). After 5 h of incubation, the radioactivity was counted and the percentage of specific lysis was calculated (means ± standard deviations). (C) Inhibition of the cytotoxicity with Ab to HLA class I in CD28-int T cells. The fractionated CD28-int CD8 T cells (Fig. ​(Fig.4A,4A, section b) and radiolabeled autologous BLC were incubated with an anti-class I monoclonal Ab (line f), isotype-matched mouse IgG Ab (line e), or medium alone (line d). After 5 h of incubation, the radioactivity was counted and the percentage of specific lysis was calculated. (D) Cytotoxic functions against other targets. The fractionated CD28-int subsets (HLA-A11/A26 and B52/B56) were tested in triplicate for cytotoxicity against 51Cr-labeled allogeneic BLC (HLA-A2/A24 and B35/B46; line g) and K562 (line h) cells with various effector/target ratios (1). After 5 h of incubation, the radioactivity was counted and the percentage of specific lysis was calculated (means ± standard deviations).

Article Snippet: For blocking experiments, an anti-class I MAb (Clone W6/32; Cederlane, Hornby, Ontario, Canada) or isotype-matched mouse IgG antibodies were used at a final concentration of 50 μg/ml.

Techniques: Activity Assay, Transformation Assay, Incubation, Isolation, Labeling, Radioactivity, Lysis, Inhibition