Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A paradox of transcriptional and functional innate interferon responses of human intestinal enteroids to enteric virus infection

doi: 10.1073/pnas.1615422114

Figure Lengend Snippet: Analysis of IFN protein from HIEs

Article Snippet: The type III IFN receptor was blocked with a polyclonal sheep antibody (25 μg/mL) against the IL28RA subunit (cat. no. AF5260, lot CCXR01; R&D Systems).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A paradox of transcriptional and functional innate interferon responses of human intestinal enteroids to enteric virus infection

doi: 10.1073/pnas.1615422114

Figure Lengend Snippet: IFN response to inactivated HRV. HIEs (j3) were inoculated with replication-competent HRV (MOI of 10 FFU) or with an equivalent amount (in micrograms) of inactivated HRV particles. (A) Expression of type III IFN (IFNL1), type I IFN (IFNB1), and the ISG IFI44L was assessed at 6 hpi by RT-qPCR. Each data bar represents the geometric mean ± 95% CI of the geometric mean (n = 4 technical replicates). Statistical analyses were performed by Mann–Whitney U test. (B) HIEs were infected as described in A and, after 1 h, HIEs were washed and resuspended in medium containing type III IFN receptor-blocking antibody (25 μg/mL anti-IL28RA). The media was analyzed at 20 hpi for secreted IFN-λ1 by ELISA. Data from one representative experiment of two independent experiments is presented as arithmetic mean ± SD of three or four technical replicates.

Article Snippet: The type III IFN receptor was blocked with a polyclonal sheep antibody (25 μg/mL) against the IL28RA subunit (cat. no. AF5260, lot CCXR01; R&D Systems).

Techniques: Expressing, Quantitative RT-PCR, MANN-WHITNEY, Infection, Blocking Assay, Enzyme-linked Immunosorbent Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A paradox of transcriptional and functional innate interferon responses of human intestinal enteroids to enteric virus infection

doi: 10.1073/pnas.1615422114

Figure Lengend Snippet: Effect of receptor-blocking antibodies on rotavirus infectivity and induction of OAS2 transcripts. (A) Stock aliquots of Ito-HRV were incubated with no antibody (control), the anti-type III IFN receptor-blocking antibody, or the anti-type I IFN system antibody mixture for 2 h. To assess the titer of HRV under these conditions, MA104 cells were infected with control Ito-HRV aliquots or antibody-treated Ito-HRV aliquots for 1 h, washed twice, and incubated overnight in DMEM for 18 h. Viral titer was subsequently obtained by FFA. (B) HIEs (j11) suspended in differentiation medium containing 0.5 mg/mL pancreatin were treated with or without blocking antibodies to the type III IFN receptor (anti-IL28RA) or the type I IFN system (anti-type 1 IFN mixture). After 1 h, 100 U/mL of IFN-λ1 or IFN-β1 was added to the HIEs. After 26 h, total RNA was extracted, and the transcriptional response was compared with HIE cultures that were neither antibody-treated nor IFN-treated. Transcript levels of the ISG OAS2 were first normalized to GAPDH levels, and the fold increase was calculated by using the 2−ΔΔCt method. Displayed above the bars is the percent reduction in OAS2 levels between the two groups being compared. Data in A and B are presented as geometric mean ± 95% CI of the geometric mean of three technical replicates. (C) HIEs (j3) were treated with or without blocking antibodies to the type III IFN receptor (anti-IL28RA) or the type I IFN system (anti-type 1 IFN mixture). After 2 h, HIEs were treated with 100 U/mL of IFN-λ1 or IFN-β1 for 24 h. After 24 h, HIEs were washed to remove the antibodies and IFN and inoculated with Ito-HRV (MOI of 0.1). The control samples consisted of HIEs which did not receive antibodies nor IFN before Ito-HRV infection. The increase in infectious virus was calculated by subtracting the titer at 1.5 hpi in the control infection from the titer at 24 hpi in each of the five test groups. Data from one representative experiment of two independent experiments is presented as arithmetic mean ± SEM of four technical replicates. Statistical analyses were performed by Mann–Whitney U test.

Article Snippet: The type III IFN receptor was blocked with a polyclonal sheep antibody (25 μg/mL) against the IL28RA subunit (cat. no. AF5260, lot CCXR01; R&D Systems).

Techniques: Blocking Assay, Infection, Incubation, Control, Virus, MANN-WHITNEY

Journal: Scientific reports

Article Title: Fusing the 3'UTR of seed storage protein genes leads to massive recombinant protein accumulation in seeds.

doi: 10.1038/s41598-023-39356-3

Figure Lengend Snippet: Figure 5. Production of IFN lambda-3 in Arabidopsis seeds. (a) Schematic representation of recombinant IFN lambda-3 fused with 3′12S1. IFN lambda-3CDS represents the coding sequence of the IFN lambda-3 gene. 6 × His- HDEL represents the ER-retention signal (HDEL) fused with a 6 × Histidine tag. (b) CBB staining of seed proteins from WT, 12s1.4, and four independent transgenic lines of IFN lambda-3–6 × His-HDEL-3′12S1/12s1.4. The arrowhead indicates IFN lambda-3 bands. (c) Immunoblot analysis of seed protein from WT, 12s1.4, and IFN lambda-3–6 × His-HDEL-3′12S1/12s1.4 plants. (d) Relative activity of purified recombinant IFN lambda-3. IFN lambda-3 activities are relative to the activity of recombinant IFN lambda-3 produced in human cells. Values are presented as the mean ± SE of four independent experiments.

Article Snippet: Monoclonal anti- IFN lambda-3 antibody (MAB5259, R&D Systems, Minneapolis, USA) was used in the immunoblot analysis.

Techniques: Recombinant, Sequencing, Staining, Transgenic Assay, Western Blot, Activity Assay, Purification, Produced

Journal: Immunity

Article Title: Human TLR-7-, -8-, and -9-Mediated Induction of IFN-α/β and -λ Is IRAK-4 Dependent and Redundant for Protective Immunity to Viruses

doi: 10.1016/j.immuni.2005.09.016

Figure Lengend Snippet: Production of IFN-α/β, IFN-λ, and TNF-α after Stimulation with Viruses in IRAK-4-Deficient Blood Cells PBMCs from controls and/or from IRAK-4-deficient patients P2, P3, or P7 were left unstimulated or stimulated with various intact and UV-inactivated viruses. (A) IFN-β mRNA levels were analyzed by reverse transcription and real-time quantitative PCR 24 hr after stimulation with intact viruses (see for the multiplicities of infection, moi). Means and standard deviations (SD) were calculated for the controls from three independent individuals, each tested once. The patients were each tested once. (B and C) IFN-α secretion (B) and IFN-λ secretion (C) were measured by ELISA after 24 hr of stimulation. Means and standard deviations (SD) were calculated for the controls from 14 independent individuals, each tested once. (D) IFN-α secretion by control PBMCs was measured by ELISA after 24 hr of stimulation by intact or UV-inactivated viruses. Means and standard deviations (SD) were calculated for the controls from seven independent individuals, each tested once. (E) TNF-α secretion was measured by ELISA after 24 hr of stimulation by two intact viruses. Means and standard deviations (SD) for controls were calculated from two independent individuals.

Article Snippet: Plates were coated with 1 μg/ml of anti-human IFN-λ mAb (AF1598, R&D Systems) overnight at 4°C, and the IFN-λ levels in the supernatant were estimated with a secondary biotinylated anti-human IFN-λ mAb (BAF1598, R&D Systems) used at a concentration of 400 μg/ml ( ).

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Infection, Enzyme-linked Immunosorbent Assay, Control

Journal: Immunity

Article Title: Human TLR-7-, -8-, and -9-Mediated Induction of IFN-α/β and -λ Is IRAK-4 Dependent and Redundant for Protective Immunity to Viruses

doi: 10.1016/j.immuni.2005.09.016

Figure Lengend Snippet: IFN-β, IFN-λ, and IL-6 Induction in IRAK-4-Deficient Fibroblasts upon Stimulation by Poly(I:C), IL-1β, TNF-α, and Viruses Induction of IFN-β, IFN-λ, and IL-6 in response to poly(I:C) (50 μg/ml), IL-1β (20 ng/ml), TNF-α (10 ng/ml), and viral stimulations in IRAK-4-deficient (P1, P2, or P4) and control fibroblasts. (A and B) IFN-β mRNA induction, as determined by Q-PCR 2 hr after stimulation (A) and IFN-β, as determined by ELISA 24 hr after stimulation (B). Means and standard deviations (SD) were calculated for each patient from two experiments, and for the controls from two independent individuals, each tested twice. (C) IFN-β and IFN-λ as determined by ELISA 24 hr after stimulation by poly(I:C), following prior treatment with IFN-α2b (10 5 U/ml for 12 hr). This experiment is representative of two independent experiments. (D) IL-6, as determined by ELISA 24 hr after stimulation. Means and standard deviations (SD) were calculated for each patient from two experiments, and for the controls from two independent individuals, each tested twice. (E) IFN-β mRNA induction 24 hr after stimulation by seven intact viruses (see for the multiplicities of infection, moi). Means and standard deviations (SD) were calculated for each patient from two experiments, and for the controls from two independent individuals, each tested twice.

Article Snippet: Plates were coated with 1 μg/ml of anti-human IFN-λ mAb (AF1598, R&D Systems) overnight at 4°C, and the IFN-λ levels in the supernatant were estimated with a secondary biotinylated anti-human IFN-λ mAb (BAF1598, R&D Systems) used at a concentration of 400 μg/ml ( ).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Infection

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