Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Soluble CD23 Controls IgE Synthesis and Homeostasis in Human B Cells i

doi: 10.4049/jimmunol.1102689

Figure Lengend Snippet: (A) Human tonsillar B cells were cultured for 8 days with IL-4 (200IU/ml) and anti-CD40 (1μg/ml) and stimulated for 30 minutes with media alone, derCD23 (3μM/48μg/ml) or triCD23 (1μM/84μg/ml). Cells were stained with mouse anti-CD21, plus secondary anti-mouse-Alexa 594 (red), anti-IgE-FITC (green), and the nuclear stain Hoescht (blue). Cells were visualized by confocal microscopy and images show a single field of view, with the bottom panel showing a three-color overlay. The white arrow indicates strong capping of mIgE with mCD21 (n=1, representative of 3). (B) Human tonsillar B cells were cultured for 12 days with IL-4/anti-CD40 alone, + anti-CD21 (0.1, 1 or 10μg/ml), + triCD23 (1μM) or + aCD21 (10μg/ml) & triCD23. IgE secretion was analysed by ELISA on day 12, relative to cells cultured with IL-4/anti-CD40 alone (n=2). (C) Cells were cultured as described in (A) and stimulated with anti-CD21 (10μg/ml), triCD23 (1μM) or anti-CD21 & triCD23. Cells were stained for mCD21 (red) and the nucleus (blue) and visualized by confocal microscopy (n=1).

Article Snippet: Goat anti-human IgE-FITC (1:500) (Vector) and monoclonal mouse anti-human IgG-APC (1:50) (Miltenyi Biotec) were added and incubated on ice in the dark for 45 minutes.

Techniques: Cell Culture, Staining, Confocal Microscopy, Enzyme-linked Immunosorbent Assay

Journal: Blood Cancer Journal

Article Title: Downregulation of PA28α induces proteasome remodeling and results in resistance to proteasome inhibitors in multiple myeloma

doi: 10.1038/s41408-020-00393-0

Figure Lengend Snippet: a Western blot analysis of ubiquitinated proteins in total protein lysates extracted from LP1 and RPMI8226 PA28α knockdown stable cells. β-actin as a loading control. b OPP pulse-chase assay of proteasome degradation and protein synthesis in LP1 PA28α knockdown stable cells. Western blot analysis of immunoglobulin lambda light chain (ƛ IgL) ( c ), eIF2α, p62/SQSTM1, and LC3B ( d ) in LP1 and RPMI8226 PA28α knockdown stable cells, β-actin as a loading control. ** P < 0.01, Student’s t test. e Working model of PA28α knockdown in MM.

Article Snippet: Antibodies used were as follows: PA28α (Cell Signaling), PSMA2 (Cell Signaling), S5a (Cell signaling), PA28β (Cell Signaling), Phospho-eIF2α (Ser51) (Cell signaling), eIF2α (Cell signaling), α-tubulin (Genetex), PA28γ (Genetex), PSMB5 (Genetex), PSMB6 (Enzo life science), PSMB7 (Genetex), PSMB8 (Genetex), PSMB9 (R&D systems), PSMB10 (R&D systems), Rpt5 (Enzo life science), Ubiquitin (Cell Signaling), β-actin (Santa Cruz Technology), TCF11/NRF1 (Cell Signaling), LC3B (Cell Signaling), p62/SQSTM1 (MBL International) human Ig lambda light chain (R&D systems), actin (Sigma). pLKO.1 empty vector, shRNA vector targeting human PA28α, NRF1 siRNA (ON-TARGETplus SMARTpool), PA28α siRNA (Accell SMARTpool), and control siRNA were purchased from Dharmacon.

Techniques: Western Blot, Knockdown, Control, Pulse Chase