EMBO/soda-nel huggingface dataset:
Intersectin-s interaction with DENND2B facilitates  recycling of epidermal growth factor receptor (2017)
Figure 2

Figure 2. DENND2B phosphorylation by PKD disrupts ITSN interaction

A HEK-293T cells were treated with DMSO or okadaic acid (OA), lysed and analyzed by Western blot.

B HEK-293T cells expressing Flag-DENND2B were treated with DMSO or 250 nM OA, and cell lysates were incubated with GST-proteins. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.

C Percentage of phosphorylated peptides following DMSO or OA treatment as determined by mass spectrometry. Low abundance peptides not included. Refer to Table EV1 for a complete peptide list.

D Alignment of DENND2B sequence from different species flanking the Ser-30 residue. PKD and 14-3-3 consensus motifs are shown on the bottom.

E HEK-293T cells expressing Flag-DENND2B were treated with DMSO, 250 nM OA or with increasing concentrations of PKD inhibitor CID755673. Cell lysates were analyzed by Western blot.

F HEK-293T cells expressing Flag-DENND2B or control cells were treated with 250 nM OA or DMSO, incubated with protein G beads coupled to anti-Flag and analyzed by Western blot.

G Lysates from HEK-293T cells expressing Flag-DENND2B and PKD-dominant negative (DN) were incubated with protein G beads or protein G beads coupled to anti-Flag. Bound proteins were detected by Western blot.

H HEK-293T cells co-expressing Flag-DENND2B with pcDNA3 or PKD-constitutively active (CA), were incubated with protein G beads or protein G beads coupled to anti-Flag and bound proteins were detected by Western blot.

I Quantification of H where PKD-CA treatment is normalized to pcDNA3 control. Mean +/- SD. Welch's t-test *p=0.016, n=6 for both treatment groups from 3 independent experiments.
Intersectin-s interaction with DENND2B facilitates  recycling of epidermal growth factor receptor (2017)
Figure 3

Figure 3. 14-3-3 outcompetes ITSN for binding to phosphorylated DENND2B

A Purified GST-14-3-3 WT or K50E were incubated with lysates of HEK-293T cells expressing GFP-DENND2B WT or S30A. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.

B HEK-293T cells expressing Flag-DENND2B were treated with DMSO or 250 nM OA and cell lysates were incubated with GST-14-3-3. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.

C Quantification of B. where bound DENND2B is normalized to the SM. Mean +/- SD. Welch's t-test **p=0.006 n=8 for both treatment groups from 5 independent experiments

D HEK-293T cells expressing Flag-DENND2B were treated with 250 nM OA alone or with 50 µm PKD inhibitor CID755673 and cell lysates were incubated with GST-14-3-3. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.

E Quantification of D. where bound DENND2B is normalized to the SM. Mean +/- SD. Welch's t-test *p=0.040. n=5 for both treatment groups from 3 independent experiments

F GST-SH3A was incubated with lysates of HEK-293T cells expressing GFP-DENND2B WT or S30A. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.

G Lysates of HEK-293T cells expressing Flag-DENND2B were pre-incubated with increasing concentrations of purified 14-3-3 WT and subsequently incubated with GST-SH3A (top panel) or GST-Grb2 (bottom panel). Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.

H Regulation of the ITSN-DENND2B interaction. In the absence of phosphorylation, DENND2B binds the SH3A (A) domain of ITSN. Phosphorylation of DENND2B by PKD occurs on Ser-30 residue within the first proline-rich domain (PRD). Phosphorylation of Ser-30 recruits 14-3-3 which outcompetes ITSN binding.
Intersectin-s interaction with DENND2B facilitates  recycling of epidermal growth factor receptor (2017)
Figure 4

Figure 4. DENND2B promotes EGFR recycling

A,B HeLa cells expressing the indicated constructs. Boxed regions are magnified on the bottom. Scale bars: 5 µm in top panel and 1 µm bottom panel. White arrows and arrowheads point to colocalization on vesicles and plasma membrane, respectively.

C Transduced MCF10A cells were quantified by real-time PCR. Mean +/- SD, ANOVA with Dunnett post-hoc test ***p=0.000 for both comparisons, n=3 from 3 independent experiments.

D Transduced HEK-293T cells expressing Flag-DENND2B were analyzed by Western blot.

E Transduced MCF10A cells, were biotinylated and cell lysates were incubated with streptavidin beads. Bound proteins were detected by Western blot.

F Quantification of E. where surface EGFR is normalized to total EGFR. Mean +/- SD, Anova with Dunnett post-hoc test *p=0.013 and ***p=0.000. n=6 for all treatment groups from 4 independent experiments

G Quantification of E where total EGFR is normalized to Hsc70. Mean +/- SD n=3 for all treatment groups from 3 independent experiments

H Schematic of trafficking assay. Surface EGFR is labelled and internalized at 16°C or recycled at 37°C. Acid washing removes labelled surface pools and allows analysis of the internal pool of EGFR originating from the surface.

I MCF10A cells expressing GFP-DENND2B were labelled for surface EGFR on ice, incubated at 16°C to allow internalization, acid washed to remove remaining labelled-EGFR and processed for immunofluorescence to visualize the internal pool of EGFR (post-endocytosis, top panel) or incubated at 37°C to allow recycling, acid washed again and processed for immunofluorescence to visualize the remaining internal pool of EGFR (post-recycling, bottom panel).

J Quantification of I top panel, internal EGFR post-endocytosis. Mean fluorescence per cell +/- SD, n=13 for GFP-DENND2B and n=21 for control cells pooled from 2 independent experiments.

K Quantification of I bottom panel, internal EGFR post-recycling. Mean fluorescence per cell +/- SD, n=10 for GFP-DENND2B and n=26 for control cells pooled from 2 independent experiments.
Intersectin-s interaction with DENND2B facilitates  recycling of epidermal growth factor receptor (2017)
Figure 5

Figure 5. EGF treatment dissociates DENND2B from ITSN

A HEK-293T cells were serum starved, treated with 100 ng/mL EGF for the indicated times and cell lysates were analyzed by Western blot.

B Quantification of A where P-PKD is normalized to Hsc70. Mean +/- SD. ANOVA with Dunnett post-test *p=0.015 and ** p=0.003 n=3 from 3 independent experiments.

C HEK-293T cells were serum starved, treated with 100 ng/mL EGF for 5 min and cell lysates were incubated with protein G beads or protein G beads coupled to anti-Flag. Bound proteins were detected by Western blot.

D Quantification of B where P-PKD substrate is normalized to Flag-DENND2B. Mean +/- SD Welch's t-test *p=0.036, n=4 from 3 independent experiments.

E HEK-293T cells were serum starved, treated with 100 ng/mL EGF for 5 min and cell lysates were incubated with GST-SH3A. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.

F Quantification of E where bound Flag-DENND2B is normalized to SM. Mean +/- SD, Welch's t-test *p=0.012, n=6 from 3 independent experiments.

G Schematic of EGFR trafficking. ITSN promotes EGFR internalization upon activation. EGF-independent activation of EGFR: EGFR traffics on Rab13-positive recycling or late (RE/LE) endosomes to the plasma membrane where ITSN binds the DENND2B to facilitate EGFR exocytosis. EGF-dependent activation of EGFR: DENND2B is phosphorylated by PKD, binds 14-3-3 and loses its affinity for ITSN and EGFR is targeted towards lysosomal degradation.

Expanded View Figure Legends
Therapeutic application of PPE2 protein of Mycobacterium tuberculosis in inhibiting tissue inflammation (2022)
Figure 3

Figure 3. PPE2 depletes mast cell population in inflamed paw tissue

(A-E) A subplantar injection of 5% of formalin (20 µl) was administered in the right hind paw of Balb/c mice and an equal volume of PBS was injected in left hind paw. After 1 hour of formalin injection, Balb/c mice were treated with a single dose of either PBS (vehicle control) or Diclofenac (10 mg/kg) or different concentrations of rPPE2 (2 mg/kg and 3 mg/kg) via intraperitoneal route and after three hours of treatment, mice were sacrificed and the paw sections were prepared and stained with Toluidine blue to quantify mast cell population. (A) Photographs of representative sections were visualized at 20X magnification (scale bar = 50 µm). (B) Counting of mast cells was performed in Toluidine blue stained paw sections using ImageJ software and were normalized per unit area (mm2). (C, D) In another experiment, subplantar injection of 5% of formalin (20 µl) was carried out in the right hind paw of Balb/c mice and equal volume of PBS was injected in another paw. After 1 hour of formalin injection, Balb/c mice were injected intraperitoneally with a single dose of either Diclofenac (10 mg/kg) or rPPE2 (3 mg/kg) or PBS (vehicle control) and after three hours of treatment, mice were sacrificed and the paw tissues were collected and analyzed for mast cell population by flow cytometry using anti-CD117 Ab and anti-FcεRI Ab (C) and percentage population of mast cells was estimated from flow cytometry (D). (E) Photographs of representative tissue sections stained with Toluidine blue were visualized at 40X magnification to observe mast cell degranulation (scale bar = 100 µm). The arrow indicates the morphology (degranulation) of mast cells.

(F-H) (F) Lysates from paw tissue were prepared and analyzed for β-hexosaminidase activity at 405 nm optical density (OD). Also, paw tissues from all groups were collected and used for cDNA synthesis, qPCR was performed to observe transcription levels of MCP-3 (G) and Mcpt4 (H). GAPDH transcript levels were used as control.

Data information: Data shown are Mean ± SEM of 8 mice. For (B, D, F-H), unpaired t-test was applied to calculate p values.
Therapeutic application of PPE2 protein of Mycobacterium tuberculosis in inhibiting tissue inflammation (2022)
Figure 5

Figure 5. PPE2 localizes to fibroblast nucleus

(A) A subplantar injection of 5% of formalin (20 µl) was done in the right hind paw of Balb/c mice and an equal volume of PBS was injected in left hind paw. After 1 hour of formalin injection, mice were administered with either PBS (vehicle control) or rPPE2 (3 mg/kg, single dose) via the intraperitoneal route. After 1 hour of rPPE2 treatment, mice were sacrificed and the paw sections were prepared. Next, paw tissue samples were deparaffinized, subjected to antigen retrieval, and were stained with anti-PPE2 Ab, and observed under a confocal microscope. Propidium iodide (PI) was used to stain the nucleus. (scale bar = 20 µm)

(B-E) In another experiment, NIH-3T3 cells (B, C) or MEFs (D, E) were incubated with either PBS or rPPE2 (3 µg/ml). After 45 minutes of rPPE2 treatment, cells were fixed, permeabilized, and stained with anti-PPE2 Ab. DAPI was used to stain the nucleus. Nuclear localization of rPPE2 in NIH-3T3 (C) and MEFs (E) was measured by calculating the colocalization coefficient.

(F-G) (F) NIH-3T3 cells were transiently transfected with either pEGFP or pEGFP-PPE2. After 24 hours of transfection, cells were fixed and observed under a confocal microscope. DAPI was used to stain the nucleus. (G) Nuclear localization of rPPE2 was measured by calculating colocalization coefficient.

Data information: Data represent Mean ± SEM of more than 50 cells of three independent experiments. For Figure C, E and G, unpaired t-test was applied to calculate p values.
Therapeutic application of PPE2 protein of Mycobacterium tuberculosis in inhibiting tissue inflammation (2022)
Figure 6

Figure 6. PPE2 inhibits scf transcription by binding to scf promoter

(A) A subplantar injection of 5% of formalin (20 µl) was done in the right hind paw of Balb/c mice and an equal volume of PBS was injected in left hind paw. After 1 hour of formalin injection, mice were administered with either a single dose of Diclofenac (10 mg/kg) or rPPE2 (3 mg/kg) or PBS (vehicle control) via intraperitoneal route. After 3 hours, paw tissues were harvested and used for cDNA synthesis. Next, qPCR was performed to observe transcription levels of SCF. GAPDH transcript level was used as an internal control. Data shown here are Mean ± SEM of 8 mice

(B) NIH-3T3 cells were activated by LPS (1 µg/ml) and after 30 minutes, cells were treated with rPPE2 (3 µg/ml) for 3 hours. Cells were then harvested for cDNA synthesis and qPCR was performed to observe transcription levels of SCF. GAPDH transcript level was used as an internal control. Data shown are Mean ± SEM of 3 independent experiments.

(C) NIH-3T3 cells were co-transfected with pGL3-SCF and pEGFP. After 24 hours of transfection, cells were treated with rPPE2 (3 µg/ml)/PBS. The control group received the empty vector (pGL3) alone. After 3 hours of treatment with rPPE2/PBS, cells were harvested and analyzed for luciferase activity. Data shown are Mean ± SEM of 3 independent experiments.

(D) Lysates prepared from the same samples used in C were resolved on SDS-PAGE and checked for GFP expression by Western blotting using anti-GFP Ab. GAPDH was used as a loading control.

(E) Varying concentrations of rPPE2 protein were incubated with [γ-32P]-ATP oligonucleotides of 60 bp spanning SCF promoter. DNA-protein complexes were resolved on 7% native polyacrylamide gel. In cold competition reactions, equimolar cold/unlabeled 60 bp oligonucleotides were used (arrow represents PPE2-oligo complex). The data shown are representative of 3 independent experiments.

Data information: For A-C, unpaired t-test was applied to calculate p values.
ApoE4 upregulates the activity of mitochondria-associated ER membranes (2015)
Figure 1

Figure 1. Phospholipid synthesis in ApoE ACM-treated cells.

A Experimental scheme. See text for details.

B Phospholipid transport/synthesis, as measured by 3H-serine incorporation, in human fibroblasts (mean±SE; n=9, with 5 replicates/experiment) and in primary mouse hippocampal neurons (mean±SE; n=3, with 4 or 5 replicates/experiment). *, p<0.05 vs E3.

C Comparison of 3H-serine incorporation in ApoE ACM-treated WT- and Mfn2-KO MEFs (mean±SE; n=3, with 3 replicates/experiment).

D Duramycin sensitivity in ApoE ACM-treated fibroblasts. Note increased sensitivity after 20 min to 5 µM duramycin in ApoE4-treated cells, which was blocked upon the addition of 15 µM exogenous PtdEtn (mean±SD; n=3, with 3 replicates/experiment).
ApoE4 upregulates the activity of mitochondria-associated ER membranes (2015)
Figure 2

Figure 2. Effect of lipoprotein-rich and -poor fractions on phospholipid synthesis.

A Experimental scheme. See text for details.

B Fractions from the discontinuous gradient were blotted with anti-ApoE (WUE-4) antibody; the doublet is a characteristic feature on ApoE western blots due to ApoE sialylation [16].

C An ApoE-containing fraction (fraction 5 [+]), an ApoE-negative fraction (fraction 3 [-]), and recombinant lipid-free ApoE protein were applied to human fibroblasts. Note increase in phospholipid synthesis using an ApoE4-containing lipoprotein fraction, but not with either an ApoE4-negative fraction or with lipoprotein-free recombinant ApoE3 or ApoE4 protein (mean ± SE; n=3, with 3 replicates/experiment).
ApoE4 upregulates the activity of mitochondria-associated ER membranes (2015)
Figure 3

Figure 3. Cholesteryl ester production in ApoE ACM-treated cells.

A Cholesteryl ester synthesis, as measured by 3H-oleate incorporation. Note increased CE in ApoE4-treated cells, whereas there was essentially no difference in triglyceride (TAG) production (mean±SE; n=3. with 3 replicates/experiment). †, p=0.06 vs E3.

B Comparison of 3H-oleate incorporation in ApoE ACM-treated WT- and Mfn2-KO MEFs (mean±SE; n=3, with 5 replicates/experiment).

C ApoE ACM-treated fibroblasts were stained with LipidTox green neutral lipid stain, and lipid droplets were visualized (example at left) and counted (right). Note significantly more lipid droplets in ApoE4-treated cells (mean ± SD, n=3).
ApoE4 upregulates the activity of mitochondria-associated ER membranes (2015)
Figure 4

Figure 4. ER-mitochondrial colocalization in ApoE ACM-treated cells.

HeLa cells were transfected with plasmids to visualize ER (in green) and mitochondria (in red), and then were treated with ApoE3- and ApoE4-ACM for 24 hours. Shown is a box-and-whisker plot comparing co-localization in ApoE3 ACM (n = 15 images analyzed [gray circles, average of co-localization in ~5 cells in each image field]) compared to that in ApoE4 ACM (n = 18 images).
Glutathione peroxidase 8 negatively regulates caspase-4/11 to protect against colitis (2019)
Figure 1

Figure 1 - GPx8-/- mice exhibit exacerbated colitis and reduced microbiome richness and diversity in the gut.

A. The design of DSS-induced colitis model for the survival experiment and studies for collection of colon samples. Mice were supplied with drinking water containing 4% DSS for 5 or 6 days, followed by 8-9 days of normal water. Colonic and blood samples were collected on day 14.

B-D. Mice were analyzed by Kaplan-Meier survival plot (B), the percentage of body weight (C), and the clinical scores (D) of WT and GPx8-/- mice with colitis induced by 4% DSS for 6 days (n = 10 per group).

E-H. Colons treated with 4% DSS for 5 days were collected on day 14. For E, F and G, 5-6 mice were used for each experiment. (E) Images and statistical analysis of colon length. (F) Images and semiquantitative scoring of hematoxylin and eosin staining of colon sections. The infiltrated immune cells at the transmural extensions were indicated by arrowhead. (G) Colon sections were stained with markers for macrophages (F4/80) and anti-GPx8. (H) Production of IL-1β, IL-6 and IL-18 in colon tissue lysates (n = 10 per group).

I, J and K. Gut microbiome was shaped by Gpx8 deficiency under standard housing conditions. Bray Curtis dissimilarity of fecal microbiota were measured by bacterial 16S RNA sequencing (n = 5 per group) and are presented by a heat map, (I), and principal coordinates analysis, (J). (K) α-diversity indicated bacteria species in each mouse depicted by richness (total operational taxonomic units, OTUs), the Chao1 richness estimator (Chao1), and the abundance-based coverage estimator (ACE) metrics.

Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).
Glutathione peroxidase 8 negatively regulates caspase-4/11 to protect against colitis (2019)
Figure 2

Figure 2 - GPx8-deficienct macrophages enhance DSS-induced colitis.

A. Illustration showing the experimental protocol for (B-H).

B. Colons from mice treated with clodronate or control liposomes and subjected to adoptive cell transfer after macrophage depletion were stained with markers for macrophages (F4/80). C-H. After undergoing macrophage depletion, C57BL/6 mice received macrophages derived from WT (WT BMDMs) or GPx8-/- mice (GPx8-/- BMDMs) were under DSS-induced colitis (n = 6-9 per group). (C) The percentage of body weight. (D) The clinical scores of mice. (E-H) Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model. (E) Statistical analysis of colon length. (F) Images and semiquantitative scoring of hematoxylin and eosin staining colon sections. (G) Colon sections stained with anti-F4/80. (H) Production of IL-1β, IL-6 and IL-18 in colon tissue lysates.

Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's one‐tailed t‐test).
Glutathione peroxidase 8 negatively regulates caspase-4/11 to protect against colitis (2019)
Figure 3

Figure 3 - Enhancement of caspase-11 activation and increased susceptibility to endotoxic shock are associated with GPx8 deficiency.

A. Canonical NLRP3 inflammasome activation was not altered by GPx8 deficiency. BMDMs were primed for 6 h with 0.5 μg/ml LPS and then transfected with LPS (TF LPS) for 8 h, stimulated with ATP (5 mM) for 3 h, MSU (150 μg/mL) for 8 h, or nigericin (5 μM) for 3 h. Activation of inflammasome was demonstrated by secreted IL-1β.

B. GPx8 deficiency enhanced noncanonical inflammasome activation. Activation of the noncanonical inflammasome by different amounts of cytoplasmic LPS was verified by IL-1β (left panel) and LDH release (right panel).

C. Upregulation of noncanonical inflammasome activation by GPx8 deficiency occurred independently of TLRs. BMDMs were primed for 6 h with 1 μg/ml poly(I:C) and then transfected with LPS for 8 h.

D, E. Over-activation of noncanonical inflammasome induced by GPx8 deficiency in BMDMs can be rescued by ectopically-expressing human GPx8 (hGPx8). (D) Activation of noncanonical inflammasome in BMDMs with empty vector (EV) or hGPx8 was verified by IL-1β (left panel) and LDH release (right panel). (E) Expression of hGPx8 mRNA in BMDMs was confirmed by quantitative reverse transcriptase PCR.

F. GPx8 deficiency enhanced caspase-11 cleavage and activation. Levels of processed caspase-1 and -11 in the supernatants (Sup) and full-length caspase-1, -11 and GAPDH in the cell lysates (Cell ext) were determined by immunoblotting. Fold changes of protein expression were normalized with internal controls and are indicated below the blots.

G. RNA expression of caspase-11 was not altered by GPx8 deficiency. Caspase-11 RNA expression of BMDMs isolated from GPx8-/- mice was measured after LPS priming.

H. GPx8 deficiency enhanced noncanonical inflammasome activation and pyroptosis in vivo. Colon samples from 5 WT or GPx8-/- mice treated with DSS for 5 days were collected on day 7. Levels of full-length and processed caspase-1, -11 and gasdermin D (GSDMD) in tissue lysates were individually analyzed by immunoblots.

I-K. GPx8-/- mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome. (I) Survival of mice primed with ultra pure LPS (E. coli O55:B5, 400 μg/kg) and re-challenged 6 h later with LPS (40 mg/kg) (n = 8 per group). (J) Survival of mice primed with poly(I:C) (LMW 1 mg/kg) and re-challenged 6 h later with LPS (10 mg/kg) (n = 9 per group). (K) IL-1β production levels in mice sera were measured 2 h after being primed with poly(I:C) (LMW 1 mg/kg) and re-challenged 6 h later with LPS (1 mg/kg) (n = 8-9 per group).

Data information: In (A-E and G), data are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats. In (K), data are presented as the mean ± SEM. NS, no significance. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).
Glutathione peroxidase 8 negatively regulates caspase-4/11 to protect against colitis (2019)
Figure 5

Figure 5 - GPx8 inhibits the activity of caspase-4 by covalently binding to cysteine 118. A. Casp4C118S mutant partially abolished the covalent disulfide-link with GPx8. Interacting GPx8 were co-immunoprecipitated with FLAG-tagged caspase-4 (WT, C118S, C329S mutants) and analyzed under reducing (+DTT) or non-reducing (-DTT) conditions. Disulfide-linked complexes of GPx8 and caspase-4 are indicated. Fold change of complexes was normalized with internal controls and indicated below blots.

B-D. C118 of caspase-4 was required for the GPx8-mediated inhibitory effect. THP-1 cells stably expressing empty vector (EV), FLAG-tagged caspase-4 (WT, C258S, C118S mutants), or GPx8 (WT and C79S mutants) were confirmed by immunoblotting (B). (C-D) Casp4C118S completely abolished the inhibition of noncanonical inflammasome signaling in response to intracellular LPS. THP-1 cells were differentiated, primed, and transfected with LPS. Activation of the noncanonical inflammasome by different quantities of cytoplasmic LPS is confirmed by the production of IL-1β (C) and LDH (D).

Data information: All data shown are representative of at least 3 independent experiments. In C and D, data are presented as the mean ± SD, n=4, technical repeats. ***P < 0.001 (Student's two‐tailed t‐test).