Fig 2: p38 interacts with and phosphorylates TFEB at serine 211. A Lysates from mouse brain were used for IP with anti-TFEB antibody or anti-p38 antibody. B, C Cell lysates from BV2 cells were used for IP with anti-TFEB antibody. SB203580 reduced the TFEB/p38 interaction. *p < 0.05. D, E Cell lysates from BV2 cells were used for IP with anti-p38 antibody. SB203580 reduced the TFEB/p38 interaction and shown in E. Mean ± SEM, n = 3. *p < 0.05. F, G Cell lysates from BV2 cells were used for IP with anti-GFP antibody. SB203580 reduced the TFEB–GFP/14-3-3 interaction. Mean ± SEM, n = 3. *p < 0.05 (H, I). Subcellular localization of TFEB was analyzed by confocal microscopy and shown in I. Scale bars, 10 μm. Mean ± SEM, n = 3, *p < 0.05. J, K BV2 cells were labeled with lysosome tracker and visualized lysosome biogenesis under a microscope and shown in K. Scale bars, 100 μm. Mean ± SEM, n = 10, *p < 0.05. L, M Cell lysates from BV2 cells were immunoblotted to determine the levels of NLRP3, ASC, cleaved CASP1, CathB and shown in M. Mean ± SEM, n = 3, *p < 0.05

Fig 3: Phosphorylation of S621 enhances RAF1 binding to 14-3-3 during HCMV infection. (A) Overexpression of Flag-RAF1-WT (wild type) or Flag-RAF1-S621A via lentiviral transduction were compared to control MRC5 fibroblasts and assessed for protein expression by western blot using antibodies specific to Flag, RAF1, and S621- or S338-phosphorylated RAF1 and total or phospho-MEK1/2 and -ERK1/2 (arrows indicate doublet). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control (n = 1). (B) Empty vector (EV), Flag-RAF1-WT, and -S621A transduced cells were mock (M)- or HCMV (H)-infected with AD169-WT at an MOI of 3.0 for 72 h in the presence or absence of 5 µM Compound C (CC) prior to Flag pulldown and co-precipitation of endogenous 14-3-3. Numbers indicate the ratio of 14-3-3 to Flag protein pulled down in the representative blot. (C) Ratios of 14-3-3 to Flag pulled down during HCMV infection lanes in (B) were quantified along with a second blot (not shown) (avg, n = 2). (D) Cells were infected at an MOI of 3.0 for 120 h or (E) at an MOI of 0.01 for 9 days (avg ±SEM, n = 4), and viral titers were assessed by TCID50. Significance was established by a one-way analysis of variance test, followed by Tukey’s multiple-comparisons test.

Fig 4: IP‐coupled mass spectrometry proteomics approach identifies novel protein–protein interactions (PPIs) of Sstr4 in keratinocytes. (A) Schematic of the co‐immunoprecipitation (Co‐IP) and mass spectrometry (MS) workflow. Cell lysates from HA‐tagged Sstr4‐ORF keratinocyte lines were incubated with anti‐HA magnetic beads to pull down interacting protein complexes. Eluted complexes were subjected to LC–MS/MS for identification of potential Sstr4 interactors. (B) Western blot analysis of whole cell lysates from the Sstr4‐ORF cell line prior to Co‐IP (n = 1). (C) Western blot validation of immunoprecipitated lysates confirming successful pulldown of Sstr4 and associated protein complexes from the Sstr4‐ORF cell line. (D) List of top protein interactors with Sstr4 in mouse keratinocytes as identified from IP‐Mass spectrometry (n = 2). (E) Protein–protein interaction network of Sstr4‐specific interactors identified from Co‐IP MS. STRING database analysis showed that 14 of these proteins form two discrete interaction networks, suggesting several coordinated and interconnected functional roles. Network made using cytoscape v3.10.3. Color around the circle corresponds to the legend. Raw data provided in Table S3. (F) Reactome pathway enrichment analysis: The 14 high‐confidence interactors forming the STRING network showed enrichment of pathways that are involved in cell cycle regulation and MAPK signaling. Font color corresponds to the specific protein in STRING network. (G) HEK293 cell lysates expressing HA‐Sstr4 or empty vector were subjected to immunoprecipitation (IP) with anti‐HA beads, followed by immunoblotting (IB) with anti‐14‐3‐3 (E9S9M) and anti‐HA antibodies. Input samples (5% total lysate) served as loading controls. The specific 14–3‐3 band (approx. 29 kDa) is enriched in the HA‐Sstr4 immunoprecipitate compared to the control (n = 2). Raw blots provided in Fig. S2.