Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Natterin bridges IFN-φ1 and non-canonical inflammasome pathways via CRFB1 /Gbp4 to license Caspy2-mediated antibacterial immunity

doi: 10.3389/fcimb.2025.1686758

Figure Lengend Snippet: Effectiveness of CRISPR/Cas9 depletion of loc795232. Basal expression of loc795232 mRNA transcripts in unstimulated WT or KO embryos ( n = 100/group) at 1 dpf by RT-qPCR (A) or by in situ hybridization (B) . All qPCR data normalized to β-actin and expressed as fold change relative to 0h WT unstimulated control. Data represent mean ± SEM of three biological replicates. Independent groups of 1 dpf embryos previously treated or not with Pam3CSK4 or MCC950 and subjected to ST stimulation for 2h, as well as KO larvae stimulated with ST were processed for Natterin detection by WB (C) using anti-Natterin serum (62 kDa dimeric form) and anti-IgG TrueBlot HRP. The corresponding bar graph represents the protein expression levels as a percentage of the control (D) , and Ponceau S staining is visualized in the first horizontal line.

Article Snippet: The specific proteins were detected in the iBindTM Flex Western System using iBind Flex Cards (Invitrogen, #SLF2010) and the solution made with the iBind solution Kit (Invitrogen, #SLF1020) and the primary antibodies: rabbit polyclonal IgG anti-mouse caspase-1 (p-10, M-20) (#sc-514, Santa Cruz Biotechnology, Dallas, United States, 1/200, detects the immature form with 50 kDa and the mature form around 28 kDa), rabbit polyclonal IgG raised against amino acids 301–350 of caspase-11 of mouse origin (p-10 (M-50; #sc-28231; Santa Cruz Biotechnology, 1/200, detects the immature form with 50 kDa and the mature form around 28 kDa) followed by rabbit HRP-labeled anti-rabbit IgG TrueBlot second antibody (Rockland, #18-8816–33 at 1/1,000) for 3h.

Techniques: CRISPR, Expressing, Quantitative RT-PCR, In Situ Hybridization, Control, Staining

Journal: Nature Communications

Article Title: V-ATPase-dependent induction of selective autophagy

doi: 10.1038/s41467-025-63472-5

Figure Lengend Snippet: a Schematic showing the phosphosites in Rpc53. The zoomed-in sequence shown with a proposed Kns1 motif (RXXS/TP) underlined and the phosphosite boxed in gray. Two overlapping GSK3 motifs (S/TXXXS/T) direct Mck1 phosphorylation of primed substrates (blue arrows) at phosphosites boxed in gray. Adapted from Lee et al., (2012). During nitrogen starvation or rapamycin treatment, Rpc53 is hyperphosphorylated and inactivated. As a result, ribosome biogenesis is repressed. b–d SEY6210 VMA2 - AID*-9MYC cells ( pRS307-CUP1p-GFP-ATG8 ) were grown to mid-log phase in YPD (0 h), centrifuged and resuspended in YPD + 0.1% DMSO, YPD + 300 μM 3-IAA, YPD + 200 nM rapamycin, or SD-N medium for the indicated times. Three biological replicates were repeated with similar results. b RPC53-TAP cell lysates were prepared and analyzed by western blot. Rapamycin treatment was the positive control that was known to induce complete Rpc53 phosphorylation. Rpc53-TAP protein was detected with anti-PAP antibody. The upper band represents phosphorylated Rpc53 (p-Rpc53-TAP) with lower mobility on an SDS-PAGE gel. c SEY6210 RPC53-TAP and RPC53 T228A -TAP cell lysates were processed and analyzed by western blot. Gcn4 protein was detected with an anti-Gcn4 antibody. *, non-specific band. d RPC53-TAP and RPC53 T228A -TAP cells transformed with empty pRS405 plasmid and pRS405-CUP1p-TRP1-3xHA plasmid were harvested, processed, and analyzed by western blot. e Graphical model of V-ATPase-dependent autophagy. 1 A, FL-associated mutations of the V-ATPase subunits cause vacuolar deacidification. 1 B, unlike nitrogen starvation-induced autophagy, the TORC1 complex remains active in this context. 2 A, The Gcn2-Gcn4 pathway is activated by the vacuolar deacidification signal. 2B, Rpc53 is partially phosphorylated by Kns1 and Mck1 and thus inactivated. This induces a downregulation of ribosome biogenesis. 2 C, the downregulation of ribosome biogenesis releases the inhibitory effect on Gcn4 translational activation, thus further stimulating the Gcn2-Gcn4 pathway. 3 A, Trp prototrophy partially inhibits Rpc53 phosphorylation, and therefore maintains ribosome biogenesis in an active state. 3B, in addition, the Trp biosynthetic pathway fuels the intracellular NAD + pool, which inhibits V-ATPase-dependent autophagy without affecting Gcn4 induction.

Article Snippet: Antisera were from the following sources: Atg8 (1:1000) , Pgk1 (1:100,000; a generous gift from Dr. Jeremy Thorner, University of California, Berkeley), monoclonal YFP (1:5000; Clontech, 632381), antibody to PAP (1:5000; Jackson ImmunoResearch, 323–005-024), antibody to Gcn4 (1:1000; C11L34 clone, Novus, NBP2-81274), antibody to p-Ser51-Sui2 (1:1000; Cell Signaling Technology, 9721) and anti-MYC antibody (1:5000; Sigma, M4439).

Techniques: Sequencing, Phospho-proteomics, Western Blot, Positive Control, SDS Page, Transformation Assay, Plasmid Preparation, Activation Assay