EMBO/soda-nel huggingface dataset:
Extracellular Vesicles deliver Mycobacterium RNA to promote host immunity and bacterial killing (2018)
Figure 3

Figure 3. EVs Released by M.tb-infected Macrophages Restrict M.tb Replication in Host cells by Activating the M.tb RNA/RIG-I/MAVS Signaling Pathway.

(A and B) M.tb CFU in WT mouse BMMs pre-treated with EVs minus (A) or plus (B) co-treatment with IFN-ɣ. BMMs were treated with EVs from uninfected (EVs_Control) or M.tb-infected (EVs_M.tb) cells for 0, 24, 48 and 72 hr following a 1 hr M.tb infection. Mock, no EV treatment. (C-F) Immunofluorescence microscopy analysis for colocalization of M.tb (GFP) with lysosome marker Lamp-1 in WT (C and D) or Mavs-/- (E and F) mouse BMMs. Cells were pre-treated for 5 hr with EVs from uninfected or M.tb-infected macrophages plus IFN-γ, and then infected with GFP-expressing M.tb for 24 hr prior to immunostaining. (D) and (F) quantitative analysis of M.tb colocalization with Lamp-1 in WT and Mavs-/- mouse BMMs respectively. (G-J) Immunofluorescence microscopy as described above except using control siRNA-treated (G and H) or RIG-I siRNA-treated (I and J) BMMs. (H) and (J) quantitative analysis of M.tb colocalization with Lamp-1 in control siRNA-treated or RIG-I siRNA-treated mouse BMMs respectively. (K-M) M.tb CFU in infected Mavs-/- (K), control siRNA-treated (L) or RIG-I siRNA-treated (M) mouse BMMs pre-treated with IFN-ɣ plus EVs from uninfected (EVs_Control) or M.tb-infected (EVs_M.tb) BMMs. CFU was determined immediately after the 1 hr infection or 24 and 72 hr post-infection. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM (C, E, G and I). n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.
Extracellular Vesicles deliver Mycobacterium RNA to promote host immunity and bacterial killing (2018)
Figure 4

Figure 4. EVs-stimulated M.tb Phagolysosome Maturation in BMMs is dependent on using EVs Released from Macrophages Infected with M.tb-expressing SecA2.

(A) Immunofluorescence microscopy analysis for colocalization of M.tb with Lamp-1 in WT BMMs pre-treated with EVs from macrophages infected with WT, ∆secA2 or secA2-complemented (∆secA2-C) M.tb CDC 1551 strains. The cells were pre-treated with EVs supplemented with recombinant mouse IFN-γ for 5 hr and subsequently infected for 24 hr with GFP-expressing M.tb. (B) quantitative analysis for the colocalization of M.tb with Lamp-1. (C) M.tb CFU in WT BMMs pre-pretreated with recombinant mouse IFN-γ and EVs from macrophages infected with WT, ∆secA2 or secA2-complemented (∆secA2-C) M.tb CDC 1551 strains. (D) and (E) as described in (A) and (B) but liposomes containing RNA isolated from EVs released from BMMs infected with WT M.tb were also added to cells 5 hr prior to infection. (F) Similar to (C), but liposome-encapsulated RNA isolated from EVs released from BMMs infected with WT M.tb was included during EV and IFN-γ pre-treatment. Data shown in (B), (C), (E) and (F) are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM (A and D). n.s., not significant; * p < 0.05 and ** p < 0.01 by two-tailed Student's t-test.
Extracellular Vesicles deliver Mycobacterium RNA to promote host immunity and bacterial killing (2018)
Figure 5

Figure 5. EVs Released by M.tb-infected Macrophages Activate LC3-associated Phagosome Maturation in BMMs via a RIG-I/MAVS-dependent Pathway.

(A and B) Immunofluorescence microscopy and quantitative analysis for colocalization of M.tb with autophagosome marker LC3 in control siRNA -treated BMMs that were untreated or pre-treated for 5 hr with recombinant mouse IFN-γ and EVs from uninfected (EVs_Control) or M.tb-infected (EVs_M.tb) macrophages followed by a 24 hr infection with GFP-expressing M.tb. Mock, untreated. (C and D) Similar to (A), but in RIG-I siRNA-treated BMMs. . (E and F) Immunofluorescence microscopy and quantitative analysis for colocalization of M.tb with markers LC3 in WT BMMs that were left untreated or pre-treated for 5 hr with recombinant mouse IFN-γ and EVs from uninfected (EVs_Control) or M.tb-infected (EVs_M.tb) macrophages, followed by a 24 hr infection with GFP-expressing M.tb. Mock, untreated. (G and H) Similar to (E and F), but in Mavs -/- BMMs. (I - L) Immunofluorescence microscopy quantitative analysis for colocalization of M.tb with NOX2 (I and J) and p47phox (K and L) as described above in WT BMMs. (M-P) similar to ( I -L), but using Mavs -/- BMMs. NOX2 (M and N) and p47phox (O and P) Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM. n.s., not significant; * p < 0.05 , ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.
Extracellular Vesicles deliver Mycobacterium RNA to promote host immunity and bacterial killing (2018)
Figure 6

Figure 6. EVs Released by M.tb-infected BMMs Attenuate M.tb Survival in Macrophages When Combined with Moxifloxacin.

(A and B) Quantitative analysis of immunofluorescence microscopy images for colocalization of M.tb with Lamp-1 in WT (A) and Mavs -/- (B) BMMs infected with M.tb for 24 hr and then treated for an additional 24 hr with moxifloxacin (MFX) and/or EVs from M.tb-infected BMMs (EVs+MFX) in the presence of recombinant mouse IFN-ɣ. Mock, no EVs or moxifloxacin treatment; EVs_Control, EVs from uninfected BMMs. (C-E) Similar to (A), but LC3 (C), NOX2 (D) and p47phox (E) colocalization with M.tb were analyzed. (F-H) As described above except Mavs -/- BMMs were used and colocalization of M.tb with LC3 (F), NOX2 (G) and p47phox (H) were quantified. (I and J) M.tb CFU analysis in WT (I) and Mavs -/- (J) BMMs infected with M.tb for 24 hr followed by treatment with EVs, moxifloxacin or combination in the presence of IFN-ɣ for 24 and 72 hr. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).
Extracellular Vesicles deliver Mycobacterium RNA to promote host immunity and bacterial killing (2018)
Figure 7

Figure 7. EVs Released by M.tb-infected Macrophages Significantly Decreased M.tb Survival in Mice When Combined with Moxifloxacin.

(A) Schematic for EVs-based adjunctive immunotherapy and moxifloxacin-based chemotherapy in M.tb-infected mice. Representative histopathological analysis for lung sections of WT (B) and Mavs -/- (D) mice that were infected with M.tb and subsequent left untreated (Mock) or treated with EVs from uninfected BMMs (EVs_Control), EVs from M.tb-infected BMMs (EVs), moxifloxacin (MXF), or combination of EVs and MXF (EVs+MXF). M.tb CFU in the lung and spleen of WT (C) or Mavs -/- (E) mice treated with EVs, MXF or combination of both. ELISA analysis for, IFN-β, TNF-α, IL-1β and IFN-γ protein level in serum isolate from M.tb-infected WT (F) or Mavs -/- (G) mice treated with EVs, MXF or a combination of both. Data shown is representative of two independent experiments. The results in (B) - (G) are the mean ± SD (n = 4 mice per group). Scale bar, 100 μM (B and D). n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).

Expanded View Figure Legends
Interleukin-38 promotes skin tumorigenesis in an IL-1Rrp2-dependent manner (2022)
Figure 1

Figure 1. IL-38 is closely related to the occurrence and development of skin tumors.

(A) Representative micrographs of human skin sections stained with hematoxylin-eosin (H&E) (left) and anti-IL-38 antibody (right) from normal patients (n = 11) and tumors of cSCC patients (n = 13). Scale bars represent 100 μm. The graph shows the quantification of mean IL-38 expression per high-powered field in tissues.

(B) Relative expression of IL-38 in human normal tissues (n = 9) and cSCC (n = 18) were analyzed using Geo Datasets (GSE98767).

(C and D) The dorsal hair of normal C57/BL6 mice were shaved and treated with DMBA/TPA twice a week for 32 weeks to induce skin tumors. (C) Representative micrographs of mouse normal skin (n = 6) and tumor (n = 6) sections stained with anti-IL-38 antibody. The graph shows the quantification of mean IL-38 expression per high-powered field in tissues. Scale bars represent 100 μm. (D) Relative expression levels of Il-38 in normal skin (n = 5) and tumors (n = 5) of mice were quantified using qPCR.

(E) Relative expression levels of Il-38 in normal tissues (n = 9) and cSCC tissues (n = 38) of mice were analyzed using Geo Datasets (GSE63967).

(F) Representative western blot bands indicating IL-38 in mouse normal skin (n = 3) and DMBA/TPA-induced tumors (n = 3). The graph shows the quantification of mean IL-38 expression in tissues.

Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test.
Interleukin-38 promotes skin tumorigenesis in an IL-1Rrp2-dependent manner (2022)
Figure 2

Figure 2. IL-38 from keratinocyte-specific knockout suppressed the progression of DMBA/TPA-induced cSCC.

(A) Timeline of DMBA/TPA treatment in two-stage chemically induced skin tumor development protocol.

(B) Representative photographs of Il-38f/f (n = 12) and K14Cre/+-Il-38f/f mice (n = 8) treated with DMBA/TPA for 32 weeks. White triangles indicate the tumors on the back of mice.

(C and D) Average tumor number (C) and volume (D) per mouse from Il-38f/f (n = 12) and K14Cre/+-Il-38f/f mice (n = 8) treated with DMBA/TPA..

(E) Tumor bearing ratio of Il-38f/f (n = 12) and K14Cre/+-Il-38f/f mice (n = 8) treated with DMBA/TPA.

(F) Representative histological micrographs stained with hematoxylin-eosin (H&E) from tumors 32 weeks after initiation. Local micro-invasive foci (red arrow) were often observed in tumors from K14Cre/+-Il-38f/f mice (lower panel). Dashed lines indicate the basement membrane. Areas indicated by boxes in the upper pictures are shown at higher magnification in the lower pictures. Top pictures, scale bars represent 800 μm; bottom pictures, scale bars represent 100 μm.

(G) Representative micrograph sections stained with K8 from tumors 32 weeks after initiation. Top pictures, scale bars represent 700 μm; bottom pictures, scale bars represent 100 μm.

(H) Malignant conversion rate of tumors 32 weeks after initiation. a=number of carcinomas/total number of tumors. b=number of mice with carcinomas/total number of mice.

Error bars represent the mean ± SD (C) or mean ±SEM (D). All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test (C and D) or Fisher's exact test (E and H).
Interleukin-38 promotes skin tumorigenesis in an IL-1Rrp2-dependent manner (2022)
Figure 3

Figure 3. IL-38 promotes inflammation-driven tumorigenesis in vivo and in vitro.

(A) Representative immunohistochemical staining micrographs of γH2AX in the skin of Il-38f/f (n = 5) and K14Cre/+-Il-38f/f (n = 5) mice after treated with DMBA for 24h. Red triangles indicate the γH2AX+ positive cells. Scale bars represent 100 μm. The graph shows the number of γH2AX+ cells per high-powered field.

(B) Taqman qPCR analysis of Hras codon 61 mutations in DMBA-treated skin of Il-38f/f (n = 5) and K14Cre/+-Il-38f/f (n = 5) mice after treated with DMBA for 24h.

(C and D) The dorsal hair of normal C57/BL6 mice were shaved and treated with DMBA/TPA twice a week for 3 weeks to induce the skin inflammation. (C) Percentage of skin-infiltrating immune cell subsets within total live cells were determined using flow cytometry in DMBA/TPA-treated Il-38f/f (n = 5) and K14Cre/+-Il-38f/f mice (n = 5). (D) Relative expression levels of inflammatory mediators in the skin of DMBA/TPA treated Il-38f/f (n = 5) and K14Cre/+-Il-38f/f mice (n = 5) were quantified using qPCR.

(E and F) Relative expression of inflammatory cytokines in A431 cells after Il-38 overexpression (E) or knockdown (F) were detected using qPCR.

Data information:

Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test.
Interleukin-38 promotes skin tumorigenesis in an IL-1Rrp2-dependent manner (2022)
Figure 4

Figure 4. IL-38 promotes the proliferation and migration via mediating key cell cycle proteins of skin cancer cells.

(A and B) The dorsal hair of normal C57/BL6 mice were shaved and treated with DMBA/TPA twice a week for 3 weeks to induce the skin inflammation. (A) Representative micrograph sections stained with hematoxylin-eosin (H&E) from the skin of Il-38f/f (n = 5) and K14Cre/+-Il-38f/f mice (n = 5) Scale bars represent 100 μm. The graph shows average epidermal thickness. (B) Representative micrograph sections stained with anti-Ki67 antibody from the skin of Il-38f/f (n = 6) and K14Cre/+-Il-38f/f mice (n = 6). Scale bars represent 100 μm. The graph shows average numbers of Ki67+ cells per high-powered field.

(C and D) Proliferation of A431 cells (n = 5) after overexpression (C) or knockdown (D) of IL-38 was detected using cell proliferation detection reagent.

(E and F) Colony-forming ability of A431 cells after IL-38 overexpression (E) or knockdown (F) analyzed using the clone formation assay.

(G and H) Representative images of the scratch assay (left) and wound closure rate (right) of A431 cells (n = 5) after overexpression (G) or knockdown (H) of IL-38.

(I and J) Representative western blot bands indicating cyclin E and cyclin A in A431 cells after IL-38 overexpression (I) or knockdown (J).

Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test. NC, Negative control; LV, Lentivirus; Oe, Overexpression.
Interleukin-38 promotes skin tumorigenesis in an IL-1Rrp2-dependent manner (2022)
Figure 5

Figure 5. IL-38 binds to IL-1Rrp2 to activate JNK/AP-1 pathway.

(A and B) Pull-down assay of IL-38 (1-152 aa) (A) or IL-38 (20-152 aa) (B) with IL-1Rrp2-Fc, IL-1RAPL1-Fc, IL1RAcp-Fc, and IgG-Fc fusion proteins.

(C) Cell extracts prepared from 293T cells transfected with a mock mammalian expression vector (left) or an expression vector encoding IL-1Rrp2 (right) were probed using anti-phospho-JNK, anti-JNK, or anti-β-actin antibodies after stimulation with IL-38 (200 ng/ml) or IL-36β (200 ng/ml) at different times.

(D) Cell extracts prepared from 293T cells transfected with an expression vector encoding IL-1Rrp2 were probed using anti-phospho-JNK, anti-JNK, or anti-β-actin antibodies after stimulation with IL-38 at different concentrations.

(E) Schematic structures of full-length IL-1Rrp2 and its deletion mutants. Lengths are indicated in amino acids (aa). SP, signal peptide; Ig, immunoglobulin domain; TIR, TIR domain.

(F) Cell extracts prepared from 293T cells transfected with HA-tagged full-length or deletion mutants of IL-1Rrp2, were probed using anti-HA or anti-β-actin antibodies. Oe, overexpression.

(G) Cell extracts prepared from 293T cells transfected with full-length or deletion mutants of IL-1Rrp2 were probed using anti-phospho-JNK, anti-JNK, or anti-β-actin antibodies after stimulation with IL-38 (200 ng/ml) at different times.

(H) After transfection with the full-length or deletion mutants of IL-1Rrp2 overexpression plasmid, 293T cells were treated with IL-38 (200 ng/ml) for an additional 6 h and AP-1 activity was measured using a luciferase reporter assay.

Error bars represent the mean ± SD. All data are biological replicates. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test.
Interleukin-38 promotes skin tumorigenesis in an IL-1Rrp2-dependent manner (2022)
Figure 6

Figure 6. IL-38 promotes the expression of inflammatory mediators and proliferation and migration of skin tumor cells dependent on IL-1Rrp2.

(A and B) Relative expression levels of inflammatory mediators were detected using qPCR after posttranscriptional silencing of IL-1Rrp2 by RNA interference, followed by rIL-38 (200 ng/mL) stimulation (A) or IL-38 knockdown (B).

(C and D) Cell proliferation detection reagents were used to examine the effect of recombinant IL-38 (200 ng/mL) protein (C) or IL-38 knockdown (D) on A431 cell proliferation after IL-1Rrp2 interference.

(E-H) Cell cloning formation assays were used to examine the effect of recombinant IL-38 (200 ng/mL) protein stimulation (E) or IL-38 knockdown (G) on A431 cell proliferation after IL-1Rrp2 interference. Representative images of the scratch assay (left) and wound closure rate (right) of A431 cells (n = 5) treated with recombinant IL-38 (200 ng/mL) (F) or knockdown of IL-38 (H) after IL-1Rrp2 interference.

Error bars represent the mean ± SD. All data are biological replicates. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using one-way ANOVA (A, B, F and H) or Student's t test (C and D).
Interleukin-38 promotes skin tumorigenesis in an IL-1Rrp2-dependent manner (2022)
Figure 7

Figure 7. IL-38 promotes the expression of inflammatory mediators and proliferation and migration of skin tumor cells in skin tumor cells dependent on JNK.

(A and B) Relative expression levels of inflammatory mediators were detected using qPCR after posttranscriptional silencing of JNK by RNA interference, followed by rIL-38 (200 ng/mL) stimulation (A) or IL-38 knockdown (B).

(C and D) Cell proliferation detection reagents were used to examine the effect of recombinant IL-38 (200 ng/mL) protein (C) or IL-38 knockdown (D) on A431 cell proliferation after JNK interference.

(E-H) Cell cloning formation assays were used to examine the effect of recombinant IL-38 (200 ng/mL) protein stimulation (E) or IL-38 knockdown (G) on A431 cell proliferation after JNK interference. Representative images of the scratch assay (left) and wound closure rate (right) of A431 cells (n = 5) treated with recombinant IL-38 (200 ng/mL) (F) or knockdown of IL-38 (H) after JNK interference.

Error bars represent the mean ± SD. All data are biological replicates. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using one-way ANOVA (A, B, F and H) or Student's t test (C and D).

Expanded View Figure Legends
Osmostress enhances activating phosphorylation of Hog1 MAP kinase by mono-phosphorylated Pbs2 MAP2K (2020)
Figure 1

Figure 1. Phosphorylation of Hog1 by osmostress in the absence of the upstream osmosensors

A. A schematic diagram of the Hog1 MAPK signaling pathway.

B. Analyses of Hog1 phosphorylation by immunoblotting with anti-phospho-p38 (Hog1-P) and anti-Hog1 (total Hog1) antibodies. Cells of the indicated genotypes were stimulated with the indicated concentrations of NaCl for the indicated time. Strains used are TM257, KT207 and KY594-1.

C. Analyses of Hog1 phosphorylation by Phos-tag band-shift assay. Yeast strain KY594-1 was stimulated with the indicated concentrations of NaCl for 5 min. The percentages of phosphorylated Hog1 [Hog1-P (%)] were calculated as explained in Materials and Methods, and are shown beneath the panel.

D. Analyses of Hog1 phosphorylation by immunoblotting with anti-phospho-p38 (Hog1-P) and anti-Hog1 (total Hog1) antibodies. Yeast strain KT219 was transformed with the indicated STE11 mutant gene carried by a single-copy plasmid that is expressed from the STE11 promoter: vec, vector; WT, wild-type; DDD, S281D/S285D/T286D. Cells were incubated with (+) or without (-) 1 M NaCl for 5 min.

E-H. Analyses of Hog1 phosphorylation by Phos-tag band-shift assay. Yeast strains (E) KY603-3; (F) TM142; (G) TM257; and (H) FP54 were stimulated with the indicated concentrations of NaCl for 5 min.

I. Comparison of the NaCl dose-responses of Hog1 activation by various strains. Phos-tag band-shift assays shown in (C and E-H) were independently repeated three times, and average values were plotted.

Data information: (C and E-H) Representative results from three independent experiments. (I) Error bars are SEM (n=3).
Osmostress enhances activating phosphorylation of Hog1 MAP kinase by mono-phosphorylated Pbs2 MAP2K (2020)
Figure 2

Figure 2. Osmostress enhances the phosphorylation of Hog1 by Pbs2

A-C. Immunoblot analyses of Hog1 phosphorylation. Yeast strains (A) KT235; (B) KT209; and (C) KT234 were stimulated with the indicated concentrations of NaCl for 10 min, and phosphorylated Hog1 (Hog1-P) and total Hog1 in cell lysates were detected by immunoblotting. The relevant genotypes of the strains are indicated in the top row of each panel, and are schematically shown in the diagrams at left. The second row from the top indicates the genes carried by a single-copy plasmid that are expressed from their own promoters. vec, vector; WT, wild-type.

D. Phos-tag band-shift analyses of Hog1 phosphorylation. The yeast strain KT234 carrying the single-copy expression plasmid YCplac22I'-Pbs2 S514D/T518D was stimulated with the indicated concentrations of NaCl for 5 min.

E. Comparison of the NaCl dose-responses of Hog1 activation by constitutively-active Ste11-Q301P and constitutively-active Pbs2-DD. Phos-tag band-shift assays shown in (D) and Figure 1E were independently repeated three times, and the average values were plotted.

F. A scheme illustrating the step in the Hog1 MAPK cascade at which osmostress acts to enhance Hog1 phosphorylation.

Data information: (E) Error bars are SEM (n=3).
Osmostress enhances activating phosphorylation of Hog1 MAP kinase by mono-phosphorylated Pbs2 MAP2K (2020)
Figure 3

Figure 3. The Hog1 L16 domain is required for the osmotic enhancement of the Pbs2-Hog1 reaction

A. Alignment of the amino acid sequences of the CD (green) and L16 (pink) domains of yeast Hog1 and mammalian p38α. The alpha helix αL16 forms the core of the L16 domain (Wang et al., 1997).

B. Schematic diagrams of Hog1-WT and its deletion constructs used in this study.

C. Immunoblot analyses of Hog1 phosphorylation. The yeast strain KT235 was transformed with pRS416-FLAG-Hog1 (WT) or its indicated deletion derivatives. FLAG-Hog1 was immunoprecipitated (IP), and immunoblotted (IB) with anti-phospho-p38 (for Hog1-P; upper panel) or anti-FLAG (for total FLAG-Hog1; lower panel)

D-G. Phos-tag band-shift assay of Hog1 phosphorylation. Yeast strain (D-E) KT235 or (F-G) KT290 carrying the single-copy expression plasmid YCplac22I'-Pbs2 S514D/T518D was transformed with either pRS416-Hog1 (WT) or its indicated mutant derivatives, and was treated with the indicated concentrations of NaCl for 5 min. (D) and (F) show typical results, and (E) and (G) summarizes the averages of three independent experiments.

H. Phos-tag band-shift assay of Hog1 phosphorylation. The yeast strain FP4 was transformed with the single-copy expression plasmid pRS416-Hog1 (WT) or pRS416-Hog1-ΔL16, and was treated with the indicated concentrations of NaCl for 5 min. The averages of three independent experiments are shown.

Data information: (E, G, and H) Error bars are SEM (n=3).
Osmostress enhances activating phosphorylation of Hog1 MAP kinase by mono-phosphorylated Pbs2 MAP2K (2020)
Figure 4

Fig 4. Inhibition of the osmotic enhancement of the Pbs2-Hog1 reaction affects the SLN1 and SHO1 branches differently

A. Phos-tag band-shift analyses of osmostress-induced Hog1 phosphorylation. The yeast strain FP4 (hog1Δ) was transformed with the single-copy expression plasmid pRS416-Hog1-ΔL16, and was stimulated with the indicated concentrations of NaCl for 5 min.

B. Averages of three independent experiments from A were plotted. Results for Hog1-WT (from Fig 1I) are included for comparison.

C-D. Same as in (A-B), except that the yeast strain KT259 (ste11Δ hog1Δ) was used.

E-F. Same as in (A-B), except that the yeast strain KY523 (ssk2/22Δ hog1Δ) was used.

Data information: (A, C, and E) Representative results from three independent experiments. (B, D, and F) Error bars are SEM (n=3).