Journal: iScience

Article Title: Dietary carbohydrate, particularly glucose, drives B cell lymphopoiesis and function

doi: 10.1016/j.isci.2021.102835

Figure Lengend Snippet:

Article Snippet: For pS6 staining, the phosphor-S6 Ribosomal Protein (Ser240/244) (D68F8) Rabbit mAb (Cell Signaling) was used as primary, and anti-rabbit IgG AF488 (Poly4064) was used as secondary.

Techniques: Recombinant, Adjuvant, Staining, Saline, Red Blood Cell Lysis, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Software, Starch

Journal: bioRxiv

Article Title: Enhanced translation expands the endo-lysosome size and promotes antigen presentation during phagocyte activation

doi: 10.1101/260257

Figure Lengend Snippet: (a) Western blot analysis of protein puromycylation in resting and activated primary macrophages. LPS increases the amount of puromycylated proteins that is blocked by p70S6K inhibitor (LY2584702) or cycloheximide. Lane 1 are control lysates from cells not exposed to puromycin. The band indicated by arrow is a non-specific band recognized by the anti-puromycin antibody. p-S6 and β-actin were used to monitor p70S6K activity and as a loading control, respectively. (b) Normalized puromycylation signal (excluding non-specific band) normalized over β-actin signal. Data is shown as the mean ± standard deviation from three independent experiments. Statistical analysis was done with an ANOVA, where * indicates conditions that are statistically distinct from control (*p<0.05). (c) Lysosomes in primary macrophages were pre-treated with LY2584702 (LY2) followed by 2 h of LPS where indicated. Images were acquired by live-cell spinning disc confocal microscopy. Scale bar = 5 µm. (d) Lysosomal tubulation was scored for each condition as shown, where a tubule was defined as longer than 4 µm in length. Tubulation index was determined by normalizing scores to resting cells. (e) Total lysosome volume in primary macrophages treated as indicated. For d and e, shown are the mean ± standard error of the mean from 30-40 cells per condition per experiment, across three independent experiments. (f) Western blot analysis of whole cell lysates from resting and activated primary macrophages with or without LY2584702. (g) Quantification of Western blots showing the levels of LAMP1 and the V-ATPase V 1 subunits H and D, normalized to β-actin. p-S6 and total S6 blots are shown to support effectiveness of LY2584702 treatment. Shown is the mean ± standard deviation of the mean from five independent blots. For b, c and e, data was statistically analysed with ANOVA and unpaired post-hoc test (*p<0.05). For each figure with Western blots, see S1 Raw Images for original, unedited Western blots. See S5 Data for original data in Fig 5.

Article Snippet: The primary antibodies used were rabbit anti-cathepsin D, ATP6V1H, ATP6V1D (GeneTex Inc., Irvine, CA), S6 ribosomal protein, phospho Ser240/244 -S6 ribosomal protein, p70 S6 kinase, phospho Thr389 -p70 S6 kinase, 4E-BP1, phospho Thr37/46 -4E-BP, β-actin, Ha-Tag and Tata-box binding protein (TBP; Cell Signaling Technologies, Danvers, MA), all at 1:1,000.

Techniques: Western Blot, Control, Activity Assay, Standard Deviation, Confocal Microscopy

Journal: bioRxiv

Article Title: Enhanced translation expands the endo-lysosome size and promotes antigen presentation during phagocyte activation

doi: 10.1101/260257

Figure Lengend Snippet: (a) Lysosomes in resting or LPS stimulated (2 h) RAW cells stably expressing the 4E-BP1 (4Ala) phosphorylation mutant or the empty pBabe vector. Images were acquired by live-cell spinning disc confocal microscopy. Scale bar = 10 µm. (b) Lysosomal tubulation was scored for each, where a tubule was defined as longer than 4 µm in length. Tubulation index was determined by normalizing scores to resting. (c) Total lysosome volume in engineered RAW macrophages treated as indicated. For b and c, shown are the mean ± standard error of the mean from 30-40 cells per condition per experiment, across three independent experiments. (d) Western blot analysis of whole cell lysates from stable cell lines. (e) Quantification of Western blots showing the levels of LAMP1 and the V-ATPase V 1 subunits H and D, normalized to β-actin for both cell lines. Anti-HA blot demonstrates expression of 4E-BP1 4Ala . Shown is the mean ± standard deviation of the mean from 3 independent blots. For b, c and e, data was statistically analysed with ANOVA and and unpaired post-hoc test (*p<0.05). For each figure with Western blots, see S1 Raw Images for original, unedited Western blots. See S6 Data for original data in Fig 6.

Article Snippet: The primary antibodies used were rabbit anti-cathepsin D, ATP6V1H, ATP6V1D (GeneTex Inc., Irvine, CA), S6 ribosomal protein, phospho Ser240/244 -S6 ribosomal protein, p70 S6 kinase, phospho Thr389 -p70 S6 kinase, 4E-BP1, phospho Thr37/46 -4E-BP, β-actin, Ha-Tag and Tata-box binding protein (TBP; Cell Signaling Technologies, Danvers, MA), all at 1:1,000.

Techniques: Stable Transfection, Expressing, Phospho-proteomics, Mutagenesis, Plasmid Preparation, Confocal Microscopy, Western Blot, Standard Deviation

Journal: bioRxiv

Article Title: Enhanced translation expands the endo-lysosome size and promotes antigen presentation during phagocyte activation

doi: 10.1101/260257

Figure Lengend Snippet: BMDCs were incubated with Eα 52–68 peptide for 4 or 6 hours in the presence or absence LPS with or without torin1 and LY2584702. Cells were then fixed and stained with Y-Ae antibodies to detect I-A b ::Eα 52–68 complex formation, and DAPI to stain nuclei. (a) I-A b ::Eα 52–68 complexes (displayed in pseudo-colour) and DAPI (grayscale) are shown for BMDCs treated as indicated. (b, c) Anti-I-A b ::Eα 52–68 antibody signal was quantified by fluorescence intensity associated with each cell. Shown is the mean of the total fluorescence intensity of I-A b ::Eα 52–68 complexes ± standard deviation from three experiments, where 50-100 cells were quantified for each. Data was analysed using ANOVA, whereby * indicates a difference compared to unstimulated BMDCs exposed to Eα 52–68 and ** indicates a difference compared to LPS-stimulated BMDCs fed Eα 52–68 (p<0.05). Scale bar = 30 µm. Color scale: 0 – 2500 (low-high). S10 Fig show similar data for HEL presentation. (d) BMDCs were fed Eα 52–68 peptide for 4 or 6 hours in the presence or absence LPS with or without torin1 and LY2584702. Following mild fixation, APCs were co-incubated with T-cells as described in methods, to measure I-A b ::Eα 52–68 complex induced T-cell activation. T-cell secreted Il-2 was measured using an ELISA system. All data were analysed using ANOVA, whereby ** indicates a difference compared to unstimulated BMDCs exposed to Eα 52–68 (p<0.05). (e) Western blot analysis of whole cell lysates from APCs. p-S6 and β-actin were used to monitor mTOR-p70S6K signaling axis activity and as a loading control, respectively. (f) Quantification of Western blots showing the levels of MHC-II (I-A/I-E) normalized over β-actin signal. Data is shown as the mean ± standard deviation from four independent experiments. Statistical analysis was done with an ANOVA, where * and ** indicates a significant difference of 2 hour and 6 hour conditions respectively, from resting cells (p<0.05). For each figure with Western blots, see S1 Raw Images for original, unedited Western blots. See S8 Data for original data in Fig 9.

Article Snippet: The primary antibodies used were rabbit anti-cathepsin D, ATP6V1H, ATP6V1D (GeneTex Inc., Irvine, CA), S6 ribosomal protein, phospho Ser240/244 -S6 ribosomal protein, p70 S6 kinase, phospho Thr389 -p70 S6 kinase, 4E-BP1, phospho Thr37/46 -4E-BP, β-actin, Ha-Tag and Tata-box binding protein (TBP; Cell Signaling Technologies, Danvers, MA), all at 1:1,000.

Techniques: Incubation, Staining, Fluorescence, Standard Deviation, Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Activity Assay, Control