EMBO/SourceData huggingface dataset:
Figure 3Western blots for STIM1 phosphorylated at S257 or S521 in (left) contracted (+) or sham (-) rat muscle and (right) muscle from mice that underwent a treadmill running protocol (+) or remained at rest (-). Total STIM1 and 14-3-3 were used as loading controls. 14-3-3 data for mouse running was re-used from Figure 1B.Western blots for phosphorylated ACC (S79) and STIM1 (S257 or S521) in L6 rat myoblasts with siRNA-mediated knockdown of endogenous STIM1 (siSTIM1) or transfected with scramble control siRNA (Control). Cells were treated with the AMPK activator A-769662 (+) or vehicle (-) for 30 min. Total STIM1 was used to demonstrate STIM1 knockdown efficiency. Total ACC and 14-3-3 were used as loading controls.Western blots for phosphorylated ACC (S79) and STIM1 (S257 or S521) in wild type (WT) or AMPK β1/β2 knockout (AMPKβ KO) mouse endothelial fibroblasts were isolated and treated with A-769662 (+) or vehicle (-) for 30 min. AMPKβ was used to demonstrate knockout, and AMPKɑ was used to demonstrate specificity for AMPKβ. Total ACC and STIM1 were used as loading controls.Western blots for total ACC and STIM1 and phosphorylated STIM1 (S257 and S521) and ACC (S79) in samples from an in vitro kinase assay to assess the ability of AMPK to phosphorylate YFP-STIM1.Representative Western blots for phosphorylated ACC (S79) and STIM1 (S257 and S521) in response to a 15 min treatment with increasing doses of thapsigargin (.01, .1, 1 or 10 µM) or .5 mM AICAR. DMSO was used as a vehicle control. 14-3-3 was used as a loading control. n=3 independent experiments.Western blots for phosphorylated ACC (S79) and STIM1 (S257 or S521) in wild type (WT) or AMPK KO mouse endothelial fibroblasts were isolated and treated with 2 µM thapsigargin (+) or vehicle (-) for 15 min.
Figure 4Time correlated single photon counting was used to estimate the fluorescence lifetime of mClover2. The mClover2:tagRFP-T fret pair was validated in HEK E cells with the fluorophores joined by either a long rigid or short flexible linker.Time correlated single photon counting was used to estimate the fluorescence lifetime of mClover2. STIM1 OASF expressed in live L6 myoblasts before (-) or after (+) treatment with A-769662.Time correlated single photon counting was used to estimate the fluorescence lifetime of mClover2. L6 myoblasts with S257 mutated to A or E.
Figure 5L6 myoblasts were transfected with the genetic cytosolic Ca2+ sensor GCaMP6s. Cells were also transfected with non-targeted siRNA (Control) or siRNA directed at endogenous STIM1 (siSTIM1). Some cells with endogenous STIM1 knockdown were also transfected with full-length wild type (WT) or mutant (S257A, S257E or L251S) STIM1-mRuby3. Representative microscopy images of GCaMP6s signal indicating SOCE in L6 myoblasts,L6 myoblasts were transfected with the genetic cytosolic Ca2+ sensor GCaMP6s. Cells were also transfected with non-targeted siRNA (Control) or siRNA directed at endogenous STIM1 (siSTIM1). Some cells with endogenous STIM1 knockdown were also transfected with full-length wild type (WT) or mutant (S257A, S257E or L251S) STIM1-mRuby3. Ca2+ levels over time (fold of 0 mM Ca2+ + EGTA basal) and (C) quantification of SOCE in in L6 myoblasts incubated in buffer containing 1 or 0 mM Ca2+ and treated with 2 µM thapsigargin during the indicated times.
Figure 6Negative geotaxis climbing assays in Drosophila (flies). Time required for 50% of flies to climb to the target line before or after 2.5 hr of continuous climbing exercise (A) in the yw strain with muscle-specific STIM knockdown using two independent RNAi sequences (RNAi26-1 or RNAi8-4) compared to GFP-expressing controls, or (B) in the w1118 strain with muscle-specific STIM knockdown using RNAiGD16187 compared to non-targeted RNAiGD12145 controls.Time to fatigue, when flies were no longer able to climb, during a 3-min period after negative geotaxis stimulation in STIM whole-body knockout flies overexpressing wild type (WT) or mutant (S257A or S257E) human STIM1-mRuby3.
Figure 1(A) Coimmunoprecipitation of SMO-HA and Myc-Smaug proteins. Extracts of Cl8 cells expressing combinations of SMOWT-HA or SMOPKA-SD-HA with Myc-SmaugWT in the absence or presence of HH were immunoprecipitated (IP) with anti-HA. The input (lower panel) and the IP complexes (upper panel) were analyzed by western blot with anti-Myc (αMyc) or anti-HA (αHA) antibodies. Here and in the other figures, the names of the proteins detected are indicated on the left and the molecular weights on the right, in kDa; the samples loaded for the input and the supernatants are equivalent to a tenth of the volume loaded for the IPs. * corresponds to higher molecular weight forms of SMO of unknown origin. Here and in panel F, the black arrow indicates unphosphorylated SMOWT-HA and the bracket indicates the phosphorylated forms of SMOWT-HA or SMOPKA-SD.(D, E) Mapping of the Smaug interaction domain in SMO. Extracts from Cl8 cells transfected with Myc-SmaugWT and various regions of SMO fused to either HA (D) or to GFP (E) were immunoprecipitated with αMyc (D) or αGFP (E) and analyzed by western blotting with αMyc (lower panel in D and upper panel in E), αHA (D, upper panel) or αGFP (E, lower panel). In: Input; Sup: supernatant. See also Fig EV1D-F.(F) Hyperphosphorylated forms of SMO do not interact with Smaug. Left panel: Extracts of Cl8 cells expressing SMOWT-HA with or without Myc-SmaugWT, in the absence or presence of HH were IP with an αMyc antibody before analysis by western blot with αHA (upper panel) or αMyc antibodies (lower panel). Right panel: Cl8 cells expressing SNAP-SMOWT with Myc-SmaugWT in the presence of HH were extracellularly labelled with a membrane-nonpermeable fluorescent SNAP substrate before cell lysis and immunoprecipitation (IP) with anti-Myc. Labelled SNAP-SMO was directly visualized after electrophoresis. Note its presence in the IP fraction with a relative enrichment of the less phosphorylated forms compared to the hyperphosphorylated forms. See also Fig EV1G-J.(G) SMO and Smaug colocalize. Representative fluorescent images of Cl8 cells expressing GFP-SmaugWT alone (1-2) or together with SMOWT-mCherry (3-3'' and 4-4'') either without (1, 3-3") or with HH (2, 4-4"). The merge images in 3" and 4" show GFP-SmaugWT in green and SMOWT-mCh in red. The same results were seen with different fluorescent tags as well (Fig EV2B). The lack of effect of HH on GFP-SmaugWT in absence of SMOWT-mCh is likely due to limiting amounts of endogeneous SMO. A least 20 cells were assayed for each condition. In absence of HH, all co-transfected cells exhibited greater than 90% colabelled SMOWT-mCherry. Scale bar (shown in G1, identical for all panels):10μm. See also Fig EV2.
Figure 2(B, C) Smaug downregulates reporter expression. Mean values of the relative levels of SNAP-GUS reporter (SNAP-GUS/GFP-SNAP ratio, in arbitrary units) in extracts of transfected Cl8 cells in the absence (black) or presence of λN-SNAP-Smaug (red), λN-SNAP (pale grey) or SNAP-Smaug (dark grey). See Fig EV3A for the representative gel and Fig EV3B which shows that λN-SNAP-Smaug has no effect on a SNAP-GUS construct that lacks the 5BoxB. N=3 (biological replicates). See also Fig EV3A-C. Here and in D-F: SNAP-GUS/GFP-SNAP ratios were set to 1 in cells expressing λN‐SNAP-Smaug. The error bars correspond to the standard deviation of the mean (SD). Statistical analysis was done by a Kruskal-Wallis test by ranks followed by a Dunn test and p values are: black bracket <0.05, red bracket <0.01, green bracket <0.0001. Here and in D-E: 15 ng of pAct.λN-SNAP-smaug plasmid were used per transfection., E) HH/SMO reduces Smaug levels and its overall negative effect on reporter expression. Mean values of the relative levels of reporter expression (determined as above) (D) or of λN-SNAP-Smaug (ratio λN-SNAP-Smaug/GFP-SNAP) (E) in the absence of SMO constructs (red) or in the presence of SMOWT-HA (green), SMOPKA-SD-HA (blue) or SMO∆958-HA (yellow), without HH (plain boxes) or in presence of HH (striped boxes). N=6 (independent biological replicates), except for "no SMO, no HH" and "SMOWT with HH" conditions where n=10 (independent biological replicates). A representative gel is shown in Fig EV3D. Note that SMOWT-HA or HH alone had reproducible weak effects that were observed in two independent biological triplicates but were not statistically significant. See also Fig EV3E-F.(F) HH/SMO reduces Smaug's intrinsic repressive ability. A similar experiment was conducted with different amounts of λN-SNAP-smaug expressing plasmid (ranging from 0 to 50 ng) as indicated). The mean values of the relative levels of reporter (y-axis) are plotted against the relative levels of λN-SNAP-Smaug (x-axis). SNAP-GUS/GFP-SNAP ratios were set to 1 in cells expressing λN‐SNAP-Smaug in absence of SMOPKA-SD/HH. The colored area represent the extent of the values observed for the relative amounts of reporter expression obtained with (blue) and without (red) SMOPKA-SD/HH, respectively. N=3 (biological triplicates). See Fig EV3H and also a representative gel in Fig EV3G.
Figure 3(A) HH/SMO promotes slow-migrating forms of Smaug. Western blot analysis of Cl8 cells that transiently express HA-SmaugWT alone, or together with either SMOWT-GFP or SMOPKA-SD-GFP, in the presence or in absence of HH. GMAP serves as a loading control. Here and in the other panels, the antibodies used are indicated on the left. Here and in (B,C), the black arrows indicate the unphosphorylated form of HA-SmaugWT and the brackets indicate the slower migrating phosphorylated forms of HA-SmaugWT. NT: not transfected. See a phosphatase assay in Fig EV4A.(B) Smaug phosphorylation requires its interaction with SMO. Western blot analysis of Cl8 cells expressing HA-SmaugWT, in presence of HH, in combination with SMO∆958-GFP, SMOPKA-SD-GFP or SMOPKA-SD, ∆978-GFP, as indicated.(C) FU controls Smaug phosphorylation. Extracts of Cl8 cells expressing SNAP-Smaug with SMOPKA-SD FU-SD-GFP (with HH) in combination with GFP-FUWT, GAP-GFP-FUWT, Myc-FUEE or GAP-null-FUDANA (as indicated) were labeled for the SNAP tag before electrophoresis. After quantification of the gels, the percentage (%) of phosphorylated Smaug (% of phosphorylated forms of SNAP-Smaug total amounts of SNAP-Smaug) was estimated. In C to E, the mean values and (SD) for independent biological triplicates (N=3) are shown in the graph at the bottom, and the statistical analysis was done by one tailed bivariate Wilcoxon rank test. See also fig EV4C, D.(D) Phosphorylation of Smaug by activated FU requires the co-expression of SMO. Mean values of the percentage of phosphorylated Smaug in extracts of Cl8 cells that express λN-SNAP-Smaug with GAP-GFP-FUWT and SMOWT alone or together (as indicated) N=3 (billogical triplicates). Note that the effect of GAP-GFP-FUWT and SMOWT together is lower than the one observed with GAP-GFP-FUWT and SMOPKA-SD FU-SD-GFP together (in panel C). This could have multiple nonexclusive causes, including the fact that SMOWT is present at much lower levels than SMOPKA-SD FU-SD [38] and/or the involvement of another kinase. See also Fig EV4B. Note that Smaug does not coIP FU unless SMO is present see Appendix Figure S2.(E) Forcing the interaction of Smaug with activated FU leads to a SMO-independent phosphorylation of Smaug. Mean values of the percentage of phosphorylated Smaug in Cl8 cells coexpressing λN-SNAP-Smaug with GFP-FU, FU-SBR, FUEE-SBR or FUDANA-SBR, with or without HH, as indicated. N=3 (biological triplicates). See also Fig EV4C and D. Note that the levels of Smaug phosphorylation observed in presence of FUDANA-SBR are not lower than those seen with GFP-FU. This suggests an incomplete inhibition of endogenous FU, which may be due to the trapping of FUDANA-SBR by Smaug. See a representative blot in Fig EV3C and a phosphatase assay in Fig EV4D.
Figure 4(A and C) Effect of FUEE-SBR on Smaug phosphorylation and levels. Extracts of Cl8 cells transfected with λN-SNAP-smaug (25 ng) and SNAP-GUS-5BoxB (300 ng) plasmids in absence or with different amounts of FUEE expressing plasmid (ranging from 15 to 60 μg, as indicated) were analyzed by electrophoresis before quantification as above. See a reprenstive gel in Fig EV5A. The % of phosphorylated SNAP-Smaug protein (determined as in Fig 3) (A) are represented as a function of the levels of FUEE-SBR. The mean values and (SD) for independent biological triplicates (N=3) are shown in the graph at the bottom. The statistical analysis was done by one tailed bivariate Wilcoxon rank test with a p value= 0.05 (*). Here CLIP-GUS was used as an internal control.(B) Effect of FUEE-SBR on Smaug activity. The relative levels of reporter expression obtained in the same experiment as in (A-C) are represented as a function of the % of phosphorylated Smaug. Color of data points correspond to ng of transfected FUEE-SBR in A-C.(A and C) Effect of FUEE-SBR on Smaug phosphorylation and levels. Extracts of Cl8 cells transfected with λN-SNAP-smaug (25 ng) and SNAP-GUS-5BoxB (300 ng) plasmids in absence or with different amounts of FUEE expressing plasmid (ranging from 15 to 60 μg, as indicated) were analyzed by electrophoresis before quantification as above. See a reprenstive gel in Fig EV5A. The relative levels of of λN-SNAP-Smaug protein (C) (determined as in Fig 2) are represented as a function of the levels of FUEE-SBR. The mean values and (SD) for independent biological triplicates (N=3) are shown in the graph at the bottom. The statistical analysis was done by one tailed bivariate Wilcoxon rank test with a p value= 0.05 (*). Here CLIP-GUS was used as an internal control.(D) Effect of FUEE-SBR on Smaug subcellular localization.Representative fluorescent images of Cl8 cells expressing GFP-SmaugWT (1), FUEE-SBR-mCherry (2), FUDANA-SBR-mCh (3) or expressing GFP-SmaugWT together with FUEE-SBR-mCherry (4-4'') or GFP-SmaugWT together with FUDANA-SBR-mCh (5-5''). The merge images in 4" and 5" show GFP-SmaugWT in green and FUEE-SBR-mCh or FUDANA-SBR-mCh in red. (6) In presence of FUEE-SBR, 274 of a total of 294 cotransfected cells show a diffuse localization of Smaug and FU and no foci. In presence of FUDANA-SBR, 100% of the cotransfected cells (n=241) show Smaug and FU distributed in bodies and colocalized. 250 ng of plasmid were used for each construct. Scale bar (shown in D1, identical for all panels): 10μ
Figure 5(A) Effect of FUEE-SBR and HH/SMO on endogeneous Smaug target mRNAs in Cl8 cells. Relative mRNA levels estimated using semi-quantitative RT-PCR for various Smaug's targets in Cl8 cells expressing FUEE-SBR (black) , FUEE (dark grey) , or FUDANA-SBR (light grey) were reported with respect to levels of a GFP control. mRNA levels were set to 1 for the GFP control. The list of genes and the sequences of the primers used are shown in Appendix Table S2 and S3. See also Fig EV5E. Here and in Fig EV5D: RNA encoding mitochondrial proteins are written in blue. N=3 (biological triplicates) and the quantifications were repeated 3 times on each sample.The results are presented as the means ± standard error. The differences between groups were assessed using Student's t-tests with Prism V8.2.1 software. A p value <0.05 was considered to indicate a statistically significant difference(B) Effect of HH on endogeneous targets of Smaug in the wing imaginal disc. Relative levels of Smaug's targets mRNAs in MS1096; UAS-hh or hhts and their respective controls (as in Fig EV5X). N=3, with xx disc for each phenotype.(C) Effect of smaug mutation on [fu1] wing class distribution. Percent distribution of phenotypic classes (defined in Appendix Figure S4) in fu1 males in presence of zero (fu1), one (fu1;;smaug47/+) or two copies (fu1;; smaug47/smaug47) of the smaug47 allele. The wings were double-blindly classified in "weak" and "strong" according to the LV3-V4 defects (see Fig EV6B). *Chi2 statistical analysis gave a p of 0,015 for fu1 and fu1;;smaug47/+ distributions and of 0,0024 when comparing fu1;;smaug47/+ to fu1;; smaug47/smaug47.
Figure 1C) MiNA analysis of HeLa cells stained with the membrane potential-independent dye MitoTracker™Green (MTG, 100 nM) to visualize and determine the mitochondrial area (= mitochondrial footprint). Left: HeLa WT cells grown in 10 mM galactose stained with 7 nM TMRE (red) plus 100 nM MTG (green). Co-localization shown in yellow. Right: mitochondrial footprint (white) and mitochondrial skeleton (red) generated from MiNA analysis of the MTG channel (green). D) Left: HeLa IF1-KO cells grown in 10 mM galactose stained with 7 nM TMRE (red) plus 100 nM MTG (green). Right: mitochondrial footprint (white) and mitochondrial skeleton (red) generated from MiNA analysis of the MTG channel (green). E) Left: HeLa IF1-H49K cells grown in 10 mM galactose stained with 7 nM TMRE (red) plus 100 nM MTG (green). Right: mitochondrial footprint (white) and mitochondrial skeleton (red) generated from MiNA analysis of the MTG channel (green). Co-localization shown in yellow. Scale bars = 10 µm. F)
Figure 2A) BN-PAGE and subsequent immuno-blotting of mitochondrial extracts from indicated HeLa cell lines showing the assembly of F1FO ATP synthase and binding of IF1. Monomers (M), dimers (D) and oligomers (O) were detected. Left panel: Coomassie-stained gel. Right panels: Immunoblotting and detection of ATP synthase and IF1. WT: wild type HeLa cells; IF*: cells expressing IF1-H49K. Stars indicate oligomeric IF1 forms. I: complex I; III: complex III.D) Assembly of sEcGFP-tagged subunits CoxVIIIa-sEcGFP (CIV#) into complex IV, and assembly of subunits γ-sEcGFP (SUγ#) and SU e-sEcGFP (Sue#) into ATP synthase. Proteins were separated by BN-PAGE and immunoblotted. Lanes #1-#10 were cut into three pieces for further evaluation. Lanes 1-2: complex IV assembly in WT and CoxVIIIa-sEcGFP-expressing cells, detection of CIV with anti-Cox1. Lanes 3-4: complex V assembly in WT and SUγ-sEcGFP-expressing cells, detection with anti-SU γ. Lanes 8-10: assembly of CIV# into CIV, SU γ-sEcGFP (SUγ#) into CV, and SU e-sEcGFP (SUe#) into CV. At lane #6 in the Coomassie-stained gel, the positions of molecular weight markers (M) are shown. SC: respiratory supercomplexes; CIV2: complex IV dimers; CIV: complex IV monomer; s1: F1-c8 subcomplexes; s2: subcomplexes of CV containing subunits b, e and g He et al., 2018(). Blue **: dimeric F1F0 ATP synthase; blue*: monomeric F1F0 ATP synthase. Red asterisks: respiratory supercomplexes
Figure 3A,A' Trajectory maps of ATP synthase labeled at SU γ (A) and SU g (A') in single mitochondria. Insets: Detailed views of the diffusion of CV-SU γ (A) and CV-SU g (A'). Scale bars: 1 μm overview and 0.5 μm insets.
Figure 4B) Diffusion coefficients for ATP synthase under control conditions (5.6 mM glucose), and under OXPHOS conditions. The probability density function (PDF) and cumulative density function (CDF) of -log D is plotted. Number of trajectories analyzed: 8622 (Glc) and 10333 (Gal) from mitochondria in at least 5 different cells.
Figure 5C) Images showing matrix pH equilibration with extracellular pH. Shown are ratiometric fluorescence images of MPP-sEcGFP before and after addition of the uncouplers and inhibitors FCCP, nigericin and oligomycin (plus 0.002 % w/v Triton X-100 where indicated). False color images (λ511/λ464) indicate ratio values. D) Ratiometric imaging of mitochondrial pH after equilibration with extracellular pH values pH=8, pH=7.4 and pH=6.5. Scale bars = 10 µm. E
Figure 6B) Mitochondrial pH values at SU e, SU γ and in the matrix bulk in hyper-glycolytic cells (high glucose: HGlc, 25 mM). C) pH at SU e , SU γ and in the matrix in respiring cells (Gal, 10 mM). D) pH at CoxVIIIa at complex IV in the ICS before and after stimulation of the respiratory chain. Da
Figure 7A) Mitochondrial pH at CV SU e measured in HeLa WT in glycolytic (HGlc) and respiring (Gal) conditions. Where indicated, 5 µg/mL oligomycin was added to inhibit CV activity. B) Mitochondrial pH at CV SU e in IF1-KO cells. Conditions: glycolytic (HGlc) and respiring (Gal) with and without oligomycin. C) Mitochondrial pH at CV SU e in IF1-OE cells. Conditions: glycolytic (HGlc) and respiring (Gal) with and without oligomycin. D)F) Effect of inhibiting the respiratory chain at complex III (CIII) with antimycin A (AA) on the local pH at SU e in WT cells (glycolytic conditions). G) Local pH at SU e in IF1-KO cells after inhibiting CIII. H) Local pH at SU e in IF1-OE cells after inhibiting CIII. Da
Figure 8A) Mitochondrial pH at CV SU e is compared for HeLa WT, IF1-KO and IF1-H49K cells under glycolytic and respiring conditions.D) pH at respiratory complex IV at SU CoxVIIIa in the ICS measured in HeLa WT, IF1-KO and IF1-H49K cells under glycolytic and respiring conditions.J) pH measurements in mitochondria in live cells expressing sEcGFP as the pH sensor at SU γ. The pH was color coded according to respective emission ratio. The pH at SU γ in IF1-H49K-expressing cells is compared with WT cells. (Scale bar: 1 µm).
Figure 9A) Mitochondrial ATP production rates in HeLa cells the presence and absence of IF1. Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were determined with an automatic flux analyzer (Seahorse/Agilent). After monitoring basal respiration, oligomycin (1 µM), and rotenone plus antimycin A (Rot; 0.5 µM; AA, 0.5 µM) were added sequentially to determine glycolytic and mitochondrial ATP production rates. ~12 technical replicates per measurement, N=4 independent assays.
Figure 1A Cell viability as assessed by GFP expression in HEK293T cells transfected with the pRetroX TETOne3G-eGFP plasmid only (vector) or pRetroX TETOne3G-eGFP harbouring the N-terminal fragment of GSDMD. Cells were treated with the indicated concentrations of doxycycline 24 h post transfection and the percentage of GFP-positive cells was determined 16 h later by flow cytometry.B LDH release from HEK293T cells transfected with the pRetroX TETOne3G-eGFP plasmid only (vector) or pRetroX TETOne3G-eGFP harbouring the N-terminal fragment of GSDMD. At 24 h post transfection, cells were treated with the indicated concentrations of doxycycline for 8 h and the percentage of LDH release was determined. Graphs show mean and s.d. of quadruplicate wells.C-D. PI staining of and LDH release from HEK293T cells transfected pRetroX TETOne3G-eGFP harbouring the N-terminal fragment of GSDMD in the presence of osmoprotectants. At 24 h post transfection, PEGs of the indicated molecular weights were added to a final concentration of 30 mM, cells were treated with 250 ng/ml doxycycline for 8 h and the level of PI staining (C) or LDH release (D) was determined.E-F. PI staining of and LDH release from LPS-primed primary BMDMs infected with log-phase S. typhimurium for the indicated timepoints in the presence of PEGs of the indicated molecular weight (numbers on the X-axis, 30 mM final concentration).* p<0.05 as determined by student's t test. Graphs show mean and s.d. of quadruplicate wells. Data are representative of at least three independent expeirments.
Figure 2A. Immunoblot analysis of cleaved GSDMD in culture supernatants and full length GSDMD, cleaved GSDMD and α-tubulin in the cell lysates of immortalized LPS-primed WT macrophages left uninfected (NS) or infected for 10-40 minutes with log phase S. typhimurium (MOI=50).C. Fractionation and immunoblot analysis for GSDMD, Na+K+ ATPase, VDAC (Voltage-dependent anion channel), HDAC1 (Histone deacetylase 1) and GAPDH (Glycerinaldehyd-3-phosphat-Dehydrogenase) of WT macrophages infected for 10 and 20 minutes with log phase S. typhimurium (MOI=50). Fractionation was carried out as described in the Material and Methods section and equivalent amount of protein was loaded per lane.D. Extraction of cleaved GSDMD from isolated membranes of WT macrophages infected for 10 minutes with log phase S. typhimurium (MOI=50). Extraction was carried in variable conditions as described in the Material and Methods section.