Journal: eLife

Article Title: Ubiquilin1 promotes antigen-receptor mediated proliferation by eliminating mislocalized mitochondrial proteins

doi: 10.7554/elife.26435

Figure Lengend Snippet: Figure 1. Mitochondrial proteins accumulate in the cytosol in the absence of UBQLN1. (A) Uninfected BJABs, or cells infected with a lentiviral construct containing non-targeted control (NTC) or UBQLN1- shRNA (construct #1 from Figure 1—figure supplement 1) were incubated with 100 ng/mL doxycycline (+dox) or vehicle for 72 hr, following which UBQLN1 and Tubulin were detected by western blot. (B) BJAB cells stably transfected with UBQLN1- (U, open symbols) or an NTC-shRNA (N, filled symbols) were incubated with doxycycline (triangles) or vehicle (squares) for 4 days. Viable cells were measured with the Cell Titer Glo (Promega) assay in duplicate with mean ±SEM, and luminescence quantified as arbitrary units. One representative experiment of two is shown. (C) Untransfected HeLa cells, or cells stably transfected with NTC- or UBQLN1 shRNA were treated similarly to BJABs and western blotted as in Figure 1A. (D) Viable cells were measured as in (Figure 1B) for HeLa cells. (E) BJAB cells containing either an NTC- or UBQLN1 shRNA construct (#1) were cultured in the presence of 100 ng/mL doxycycline starting at day 2 and then washed to remove doxycycline (grey-shaded box). The number of viable cells was estimated by CellTiter Glo as arbitrary units of luminescence in duplicate wells. Each line represents the average of duplicate wells with mean ± SEM. (F) Isolated cytosolic protein from NTC- or UBQLN1- shRNA expressing BJABs was labeled in triplicate with TMT and analyzed by mass spectrometry. Shown are the roughly 1300 significantly altered (p<0.05) proteins and their relative enrichment Figure 1 continued on next page

Article Snippet: BJAB cells RRID:CVCL_ 5711 (originally from DSMZ) were confirmed as mycoplasma-negative and cultured in complete medium with RPMI, Pen/strep, L-glutamine (Gibco, Thermo Fisher), and 10% FBS (Sigma).

Techniques: Infection, Construct, Control, shRNA, Incubation, Western Blot, Stable Transfection, Transfection, Cell Culture, Isolation, Expressing, Labeling, Mass Spectrometry

Journal: The Journal of clinical endocrinology and metabolism

Article Title: HOXA10, Pbx2, and Meis1 protein expression in the human endometrium: formation of multimeric complexes on HOXA10 target genes.

doi: 10.1210/jc.2004-0817

Figure Lengend Snippet: FIG. 4. Meis1 is absent and Pbx2 is present in glandular epithelial Ishikawa cells. A, MEG-01 cell extract was used as a positive control for Meis1. Meis1 is absent in the Ishikawa cell extract. Goat IgG was used as a negative control antibody. B, BJAB cell extract was used as a positive control for Pbx2. Pbx2 is present in the Ishikawa cell extract. Rabbit IgG was used as a negative control antibody.

Article Snippet: BJAB cell lysate was used as a positive control for Pbx2 and megakaryoblastic cell lysate (Santa Cruz Biotechnology) was used as a positive control for Meis1.

Techniques: Positive Control, Negative Control

Journal: The Journal of Clinical Investigation

Article Title: Evolutionary conservation of human ketodeoxynonulosonic acid production is independent of sialoglycan biosynthesis

doi: 10.1172/JCI137681

Figure Lengend Snippet: A continuous cell viability assay (0–84 hours) was performed to evaluate mannose toxicity to various human and mouse cell lines including mutants of Kdn-associated metabolic enzymes. The mannose feeding concentration was set at 0–200 mM, and the same concentration of GlcNAc was used as a control for the hyperosmolarity effect. Results shown are for (A and B) HEK293A cells; (C and D) HUVECs; (E and F) BJAB K88 cells (GNE WT); (G and H) BJAB K20 cells (GNE-KO); (I and J) PMI WT cells; and (K and L) PMI-KO cells. (L) PMI-KO cells were always maintained in mannose-supplemented (25 μM) media for their survival during GlcNAC feeding. Data are representative of 2 independent experiments.

Article Snippet: HEK293A cells (Thermo Fisher Scientific) were cultivated in RPMI 1640 media with 10% FBS; HUVECs (American Type Culture Collection [ATCC]) in EBM2 media (Lonza); BJAB K20 and BJAB K88 cells (provided by Stephan Hinderlich and the late Werner Reutter, Beuth University of Applied Sciences, Berlin, Germany) in RPMI 1640 media with 10% FBS; PMI WT and PMI-KO cells in DMEM containing 1 g/L glucose and 10% FBS; and HK2 cells in keratinocyte serum-free media supplemented with bovine pituitary extract and recombinant EGF (all from Gibco, Thermo Fisher Scientific).

Techniques: Viability Assay, Concentration Assay, Control

Journal: The Journal of Clinical Investigation

Article Title: Evolutionary conservation of human ketodeoxynonulosonic acid production is independent of sialoglycan biosynthesis

doi: 10.1172/JCI137681

Figure Lengend Snippet: Levels of cytosolic free (and CMP-) Kdn (A–E) and CMP-Kdn (F–J) were measured by DMB-HPLC. Results are shown for (A and F) HEK293A cells; (B and G) BJAB K88 (GNE WT) cells; (C and H) BJAB K20 (GNE-KO) cells; (D and I) PMI WT mouse embryonic fibroblasts; and (E and J) PMI-KO cells. CMP-Sias were measured by DMB-HPLC after NaBH4 treatment. The mannose concentration was set at 0 or 15 mM, except for PMI-KO cells (0.025, 0.25, and 1 mM). Data are shown as the mean (SD). *P < 0.05, by unpaired t test.

Article Snippet: HEK293A cells (Thermo Fisher Scientific) were cultivated in RPMI 1640 media with 10% FBS; HUVECs (American Type Culture Collection [ATCC]) in EBM2 media (Lonza); BJAB K20 and BJAB K88 cells (provided by Stephan Hinderlich and the late Werner Reutter, Beuth University of Applied Sciences, Berlin, Germany) in RPMI 1640 media with 10% FBS; PMI WT and PMI-KO cells in DMEM containing 1 g/L glucose and 10% FBS; and HK2 cells in keratinocyte serum-free media supplemented with bovine pituitary extract and recombinant EGF (all from Gibco, Thermo Fisher Scientific).

Techniques: Concentration Assay

Journal: The Journal of Clinical Investigation

Article Title: Evolutionary conservation of human ketodeoxynonulosonic acid production is independent of sialoglycan biosynthesis

doi: 10.1172/JCI137681

Figure Lengend Snippet: Mouse monoclonal anti–Kdn IgG antibody, with kdn3G recognizing the (Kdn) ganglioside GM3 and mouse monoclonal anti–Kdn IgM antibody, with kdn8kdn recognizing the α2,8-linked Kdn were used for flow cytometry to analyze cell-surface expression of Kdn. Results are shown for (A and B) BJAB K20 (GNE-KO) cells and (C and D) PMI-KO cells, with or without mannose feeding. (E) Empty vector, Cmas1, and Cmas2 were transiently expressed in HEK293A cells. Cells were harvested after 36 hours of mannose feeding (0 or 15 mM), and then membrane fractions were prepared to analyze the conjugated form of Sia by DMB-HPLC analysis (Kdn, Neu5Gc, and Neu5Ac). Representative HPLC peaks for empty vector (F and G) or Cmas2-transfected (H and I) membrane samples. (F and H) 0 mM and (G and I) 15 mM mannose feeding. Data are representative of 2 independent experiments. Ac, Neu5Ac; Gc, Neu5Gc; Man, mannose.

Article Snippet: HEK293A cells (Thermo Fisher Scientific) were cultivated in RPMI 1640 media with 10% FBS; HUVECs (American Type Culture Collection [ATCC]) in EBM2 media (Lonza); BJAB K20 and BJAB K88 cells (provided by Stephan Hinderlich and the late Werner Reutter, Beuth University of Applied Sciences, Berlin, Germany) in RPMI 1640 media with 10% FBS; PMI WT and PMI-KO cells in DMEM containing 1 g/L glucose and 10% FBS; and HK2 cells in keratinocyte serum-free media supplemented with bovine pituitary extract and recombinant EGF (all from Gibco, Thermo Fisher Scientific).

Techniques: Flow Cytometry, Expressing, Plasmid Preparation, Membrane, Transfection