Journal: Viruses

Article Title: Urine and Free Immunoglobulin Light Chains as Analytes for Serodiagnosis of Hantavirus Infection

doi: 10.3390/v11090809

Figure Lengend Snippet: Western blot analysis of urine samples. Urine samples (30 µL/lane) separated in SDS-PAGE under non-reducing conditions were blotted onto nitrocellulose and sequentially immunoblotted with anti-lambda and anti-kappa light chain antibodies. Patients #1–#4 are included in . The panels on left show the results for anti-lambda light chain staining (probed first, detected using IR800-conjugated secondary antibody) and the panels on right show anti-kappa light chain staining (detected using AF680-conjugated secondary antibody). Molecular weight markers (Bio-Rad, precision plus protein dual color standards) are always the leftmost lane. FLC (free light chain) indicates monomeric and FLC 2 dimeric FLCs. All detections were performed using an Odyssey Infrared Imaging System (LI-COR Biosciences).

Article Snippet: We coupled mouse monoclonal anti-kappa (clone 4C11) and anti-lambda (clone 3D12) free light chain antibodies (both from HyTest Ltd., Turku, Finland) to Pierce NHS-activated Magnetic Beads (Thermo Fisher Scientific, Vantaa, Finland) following the manufacturer’s protocol with 400 μg of antibody per 500 μL of activated bead slurry.

Techniques: Western Blot, SDS Page, Staining, Molecular Weight, Imaging

Journal: Viruses

Article Title: Urine and Free Immunoglobulin Light Chains as Analytes for Serodiagnosis of Hantavirus Infection

doi: 10.3390/v11090809

Figure Lengend Snippet: Immunoprecipitation (IP) of PUUV N protein using FLCs and purification of free kappa light chains from urine: ( A ) Monoclonal antibodies against free kappa (clone 4C11) and lambda (3D12) light chains were conjugated to Pierce NHS-activated magnetic beads (Thermo Fisher Scientific) and used for IP of FLCs and PUUV N protein. The left lanes show anti-kappa IP and the right lanes anti-lambda IP results of AF647-labeled PUUV N protein. The samples are indicated above each lane (u stands for urine and p for plasma); the PUUV+ pools were represented by samples collected during hospitalization. The bound PUUV N protein was visualized using an Odyssey Infrared Imaging System (LI-COR Biosciences) at IR700 channel after SDS-PAGE separation; M represents the molecular weight marker (Bio-Rad, precision plus protein dual color standards); ( B ) The experimental setup described in panel A was used for IP of AF647-labeled PUUV with FLCs from the urine and plasma of healthy volunteers and PUUV patients (three time points). The left panel shows the results of IP with anti-kappa coated beads and the right panel IP with anti-lambda coated beads. The samples are indicated above each lane (u stands for urine and p for plasma) ( C ) Eluates (20 μL/lane) from monoclonal (clone 4C11) free kappa light chain antibody coupled CNBr-activated Sepharose 4B columns after passing through urine from patients with acute PUUV infection (PUUV+, 2 mL) and healthy volunteers (PUUV−, 15 mL) were analyzed by western blotting using a polyclonal anti-kappa light chain antibody. Detection was performed used an Odyssey Infrared Imaging System (LI-COR Biosciences), M represents the molecular weight marker (Bio-Rad, precision plus protein dual color standards).

Article Snippet: We coupled mouse monoclonal anti-kappa (clone 4C11) and anti-lambda (clone 3D12) free light chain antibodies (both from HyTest Ltd., Turku, Finland) to Pierce NHS-activated Magnetic Beads (Thermo Fisher Scientific, Vantaa, Finland) following the manufacturer’s protocol with 400 μg of antibody per 500 μL of activated bead slurry.

Techniques: Immunoprecipitation, Purification, Bioprocessing, Magnetic Beads, Labeling, Clinical Proteomics, Imaging, SDS Page, Molecular Weight, Marker, Infection, Western Blot

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

Journal: Scientific Reports

Article Title: Engineering a reporter cell line to mimic the high oligomannose presenting surface immunoglobulin of follicular lymphoma B cells

doi: 10.1038/s41598-020-79862-2

Figure Lengend Snippet: FACS and fluorescence in vivo imaging. ( A ) Fluorescent microscopy of engineered BZ-mCherry cell line shows the clear red fluorescence of the reporter cell line (scale bar = 50um). ( B ) Ectopic murine BZ-mCherrry tumors are clearly visible by fluorescence in vivo imaging taken at Em = 620, Ex = 580, Bin = 4/4, Fnumber = f2, exposure = 0.5 s. ( C ) The potential use of the BZ-mCherry cell line for FACS based screening is also evident by the bright red fluorescence. ( D ) We also see a large shift in the mean fluorescent intensity for the BZ-mCherry cells compared to HEK when incubated with FITC-labeled anti-lambda probe.

Article Snippet: FITC labeled anti-lambda light chain (Cat# NB7551 Novus Biologicals) antibody was diluted 1:2000 ratio and blocked with 1% BSA in PBS buffer for 1 h at room temperature with rocking (50 rpm).

Techniques: Fluorescence, In Vivo Imaging, Microscopy, Incubation, Labeling