Journal: Frontiers in Immunology

Article Title: BmSPP is a virus resistance gene in Bombyx mori

doi: 10.3389/fimmu.2024.1377270

Figure Lengend Snippet: BmNPV proliferation was inhibited after treatment of BmSPP inhibitor (Z-LL) 2 -Ketone. (A) The cell growth of BmN cells after 72 hours of incubation with (Z-LL) 2 -ketone was observed under a microscope. (B) Cell viability assay after 72 hours of drug administration. (C) After incubation with different concentrations of inhibitor, BmN cell was infected by BmNPV-GFP and observed the viral fluorescence at 0, 12, 24, and 48 hpi. (D-G) After incubation with different concentrations of inhibitors, the expression of BmNPV genes were determined by qPCR at 0, 12, 24, and 48 hpi at different infection stages, IE1 (D) , Helicase (E) , GP64 (F) , VP39 (G) . Data are given as mean ± SD (n = 3). Student’s t-tests were used for statistical analysis (ns P>0.05, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001).

Article Snippet: The SPP inhibitor ((Z-LL) 2 -ketone) (Sigma) was dissolved in DMSO.

Techniques: Incubation, Microscopy, Viability Assay, Infection, Fluorescence, Expressing

Journal: iScience

Article Title: Dynamic association of the intramembrane proteases SPPL2a/b and their substrates with tetraspanin-enriched microdomains

doi: 10.1016/j.isci.2023.107819

Figure Lengend Snippet: Intramembrane proteolysis of the CD74 NTF is impaired in CD81-deficient cells (A) CD74 was transiently expressed in HEK293 wild type (WT), CD9 KO, and CD81 KO cells. Total lysates were analyzed by Western blotting using a tris-glycine buffer system. CD74 was detected with an antibody against an N-terminal epitope detecting both the CD74 full length (FL) protein as well as derived N-terminal fragments (NTF). (B) Densitometric quantification of (A). Levels of the CD74 NTF were normalized to those of the full length protein (NTF/FL) and the resulting ratio then normalized to that of the WT cells. N = 3, n = 6. One-way ANOVA with Tukey’s post-hoc test. ∗∗, p < 0.01. (C) CD74 was transiently expressed in WT or CD81 KO HEK293 cells. As indicated, cells were co-transfected with a CD81-3xFLAG expression plasmid or empty vector (−). For detection of CD74, electrophoretic separation was performed with a tris-tricine buffer system which provides a better resolution of the individual forms and fragments of CD74. CD81 expression was also confirmed by Western blotting. (D–F) Quantification of (C). CD74 NTF/FL ratios were determined in the different samples and normalized to the level of the empty vector-transfected WT cells. Mean ± S.D. N = 4, n = 7. One-way ANOVA with Tukey’s post-hoc test. ∗, p < 0.05 (E) In order to analyze the subcellular targeting of CD74, wild type (WT) or CD81 KO HEK293 cells were transiently transfected with expression constructs of murine CD74. Following fixation, the distribution of CD74 was visualized using a polyclonal antiserum directed against the N-terminus of CD74 by indirect immunofluorescence. In parallel, the endo/lysosomal marker LAMP-2 was detected. Zoomed regions are represented by white boxes. Scale bar, 10μm or 2 μm for the digital zoom. The co-localization of CD74 and LAMP2 was analyzed using Fiji and the JACop plugin. Manders co-localization coefficient M1 was calculated which represents the percentage of above-background pixels in the images for the CD74 staining overlapping with the above-background pixels in the image for LAMP2. Each data point represents analysis of the CD74-expressing cells in one microscopic image. Mean ± S.D. N = 3, n = 13–14. One-way ANOVA with Tukey’s post-hoc test. ∗∗∗, p < 0.001 (F) CD74 was transiently expressed in WT and CD81 KO HEK293 cells. Lysates were prepared in presence of 1% Brij-98 and separated using discontinuous sucrose density gradients at 263,627 x g max for 16 h at 4°C. Thirteen fractions of 1 mL were collected starting from the top. Equal volumes of each fraction were analyzed by Western blotting with an antibody detecting the N-terminus of CD74. (G) BMDCs from wild-type mice were treated with the cysteine protease inhibitor E−64d (40 μM), a combination of (Z-LL) 2 -ketone (40 μM) and inhibitor X for (1 μM) (ZLL/InX) for inhibiting SPPL2a or DMSO as control (−) for 24 h prior to cell harvest. Lysates were subjected to Western blotting employing a tris-tricine buffer system in order to analyze processing of endogenous CD74. Different NTFs were detected depending on the applied inhibitors which are referred to as NTF1, NTF2, and NTF3 in the following. (H) BMDCs treated as described in (G) were lysed in presence of 1% Brij-98. Lysates were separated by density gradient centrifugation as in (F). Fractions were analyzed by Western blotting (tris-glycine buffer system) for presence of full length CD74 and the different NTFs. To provide better separation of the distinct NTFs, samples were analyzed in parallel by tris-tricine SDS-PAGE prior to Western blot analysis, which is shown below the tris-glycine blots. In order to allow identification of the different NTFs by correlation with (G), total lysates of BMDCs (WCL) treated with the same inhibitors were analyzed in parallel to the respective gradient fractions when performing the tris-tricine SDS-PAGE.

Article Snippet: Where indicated, BMDCs were treated with a combination of (Z-LL) 2 -ketone (40 μM, Peptanova) and inhibitor X (1 μM, Tocris) or E-64d (40 μM, Enzo).

Techniques: Western Blot, Derivative Assay, Transfection, Expressing, Plasmid Preparation, Construct, Immunofluorescence, Marker, Staining, Protease Inhibitor, Gradient Centrifugation, SDS Page

Journal: iScience

Article Title: Dynamic association of the intramembrane proteases SPPL2a/b and their substrates with tetraspanin-enriched microdomains

doi: 10.1016/j.isci.2023.107819

Figure Lengend Snippet:

Article Snippet: Where indicated, BMDCs were treated with a combination of (Z-LL) 2 -ketone (40 μM, Peptanova) and inhibitor X (1 μM, Tocris) or E-64d (40 μM, Enzo).

Techniques: Virus, Recombinant, Agarose Gel Electrophoresis, Protease Inhibitor, Modification, Western Blot, Transfection, Extraction, Bicinchoninic Acid Protein Assay, Plasmid Preparation, Software

Journal: bioRxiv

Article Title: Intramembrane protease SPP defines a cholesterol-regulated switch of the mevalonate pathway

doi: 10.1101/2021.07.19.452877

Figure Lengend Snippet: (A) Experimental outline of the SILAC-based organelle proteomics. (B) ER-resident membrane proteins quantified by organelle proteomics (mean H/L ratio, n =2). (C) Western blot (WB) showing steady state analysis of endogenous SQS and HMGCR in Hek293T cells upon 16 h treatment with (Z-LL) 2 -ketone or DMSO. Actin was used as loading control. Quantification shows Mean ± SEM, n =4. (D) Cycloheximide (CHX) chase of endogenous SQS in Hek293T cells in presence of (Z-LL) 2 -ketone or vehicle control (DMSO). Monomeric and SDS-stable dimeric forms of SPP are indicated (m and d, respectively). Actin was used as loading control. Mean ± SEM, n=3 . (E) CHX chase of ectopically expressed FLAG-tagged SQS in Hek293T cells co-expressing HA-tagged wild-type (wt) SPP and catalytic mutant SPP D265A (DA) revealing processing of the full-length form (black arrow) to an N-terminal fragment (white arrow). (F) Western blot analysis showing increased SQS levels upon tamoxifen induced SPP knock out in mice bone marrow-derived dendritic cells. Western blot and quantification show 4 biological replicates. Actin was used as loading control.

Article Snippet: For inhibition of the proteasome and SPP, 2 µM epoxomicin and 50 µM (Z-LL) 2 -ketone (Merck Millipore) were added from stock solutions in dimethyl sulfoxide (DMSO).

Techniques: Western Blot, Expressing, Mutagenesis, Knock-Out, Derivative Assay

Journal: bioRxiv

Article Title: Intramembrane protease SPP defines a cholesterol-regulated switch of the mevalonate pathway

doi: 10.1101/2021.07.19.452877

Figure Lengend Snippet: (A) Outline of TRC8 constructs used. (B) Doxycycline-induced expression of HA-tagged TRC8 wt or the Y32E mutant in Hek293ΔTRC8 cells. Cells were subjected to CHX chase and assayed for endogenous SQS. Mean ± SEM, n=3 . (C) Doxycycline-induced expression of HA-tagged TRC8 wt or RING domain mutant (RING mt ) in Hek293ΔTRC8 cells. Cells were subjected to CHX chase and assayed for endogenous SQS. Mean ± SEM, n=3 . (D) Doxycycline-induced expression of HA-tagged TRC8 wt or the RING mt in Hek293ΔTRC8 cells in cholesterol depletion or repletion. Cells were assayed for endogenous SQS by CHX chase as outlined in . Mean ± SEM, n=3 . (E) Doxycycline-induced expression of HA-tagged TRC8 RING mt or the RING mt Y32E double mutant in Hek293ΔTRC8 cells. Cells were subjected to CHX chase assay and assayed for endogenous SQS. Mean ± SEM, n=3 . (F) Doxycycline-induced expression of HA-tagged TRC8-RING mt in Hek293ΔTRC8 cells was assayed for endogenous SQS by CHX chase in presence or absence of (Z-LL) 2 -ketone (inh.). Mean ± SEM, n=4 . (G) Activity probe labelling of endogenous SPP-S-tag in microsomes isolated from cells grown in FBS or LPDS containing medium. SDS-PAGE analysis and quantification of probe binding is shown for 4 independent experiments.

Article Snippet: For inhibition of the proteasome and SPP, 2 µM epoxomicin and 50 µM (Z-LL) 2 -ketone (Merck Millipore) were added from stock solutions in dimethyl sulfoxide (DMSO).

Techniques: Construct, Expressing, Mutagenesis, Activity Assay, Isolation, SDS Page, Binding Assay