Journal: eLife

Article Title: Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20 Slp1 under stress

doi: 10.7554/eLife.97896

Figure Lengend Snippet: ( A ) Co-immunoprecipitation analysis on MCC–APC/C association. Cells with indicated genotypes were grown at 30°C to mid-log phase and arrested at 18°C for 6 hr. Lid1-TAP was immunoprecipitated and associated Mad2, Mad3, and Slp1 Cdc20 were detected by immunoblotting. The amount of co-immunoprecipitated Mad2, Mad3, and Slp1 Cdc20 was quantified by being normalized to those of total immunoprecipitated Lid1 in each sample, with the relative ratio between Mad2-GFP plus Mad3-GFP or Slp1 Cdc20 and Lid1-TAP in wild-type sample set as 1.0. Blots are representative of three independent experiments. p values were calculated against wild-type cells. ( B, C ) Immunoblot analysis of Slp1 Cdc20 abundance in nda3-KM311 cells treated at 18°C for 6 hr. Slp1 Cdc20 levels were quantified with the relative ratio between Slp1 Cdc20 and Cdc2 in wild-type strain set as 1.0. Phosphorylated Pmk1 (Pmk1-P) or phosphorylated Sty1 (Sty1-P) in ( A ) were detected using anti-phospho p42/44 and anti-phospho p38 antibodies and represents activated cell integrity pathway (CIP) or stress-activated pathway (SAP) signaling, respectively. sty1-T97A was inactivated by 5 μM 3-BrB-PP1. Blots shown are the representative of three independent experiments. p values were calculated against wild-type cells. ( D ) Real time quantitative PCR (RT-qPCR) analysis of mRNA levels of slp1 + . Cells with indicated genotypes were grown and treated as in ( A–C ) before RNA extraction. The relative fold-change ( slp1 + / act1 + ) in mRNA expression was calculated with that in wild-type cells being normalized to 1.0. Note mRNA level of slp1 + in pek1 DD mutant is not decreased. Error bars indicate mean ± standard deviation of three independent experiments. Two-tailed unpaired t -test was used to derive p values. ( E ) Schematic summary of the negative effect of activated CIP and SAP signaling on APC/C activation based on primary phenotype characterization of pmk1Δ , sty1-T97A , pek1 DD , and wis1 DD mutants. Figure 2—source data 1. Uncropped blots for . Figure 2—source data 2. Raw data of co-immunoprecipitation rate of Mad2/Mad3/Slp1, Slp1 level measurement, and RT-qPCR for . Figure 2—source data 3. Raw unedited blots for .

Article Snippet: To remove Slp1 Cdc20 phosphorylation, one of the immunoprecipitated samples was treated with 400 units of Lambda protein phosphatase (New England Biolabs, P0753S) at 30°C for 30 min. Co-immunoprecipitated Slp1 Cdc20 was detected with rabbit polyclonal anti-Slp1 antibodies, and the phosphorylated form of Slp1 Cdc20 was detected with rabbit polyclonal anti-pSer28/pThr31 antibodies, respectively.

Techniques: Immunoprecipitation, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, RNA Extraction, Expressing, Mutagenesis, Standard Deviation, Two Tailed Test, Activation Assay

Journal: eLife

Article Title: Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20 Slp1 under stress

doi: 10.7554/eLife.97896

Figure Lengend Snippet: ( A ) Schematic depiction of the arrest-and-release experiment design. For cdc25-22 background strains, cells were grown at 25°C and then arrested at the G 2 /M transition by shifting to 36°C for 3.5 hr. The cultures were released by shifting back to 25°C, and aliquots were taken at different time intervals and were fixed and stained with DAPI to monitor cell-cycle progression. ( B ) The percentages of binucleated cells were counted based on DAPI staining for each time point after release at 25°C. ( C ) Samples of indicated strains taken at 60, 75, and 90 min after being shifted back from 36 to 25°C were subjected to immunoblotting with anti-Slp1 and anti-PSTAIR antibodies to detect total Slp1 and Cdc2, respectively. Blots shown are representative of two independent biological replicates. Figure 2—figure supplement 1—source data 1. Uncropped blots for . Figure 2—figure supplement 1—source data 2. Raw data of time-course analyses of arrest-and-release of cdc25-22 mutants for . Figure 2—figure supplement 1—source data 3. Full raw unedited blot (Slp1) for . Figure 2—figure supplement 1—source data 4. Full raw unedited blot (Cdc2) for .

Article Snippet: To remove Slp1 Cdc20 phosphorylation, one of the immunoprecipitated samples was treated with 400 units of Lambda protein phosphatase (New England Biolabs, P0753S) at 30°C for 30 min. Co-immunoprecipitated Slp1 Cdc20 was detected with rabbit polyclonal anti-Slp1 antibodies, and the phosphorylated form of Slp1 Cdc20 was detected with rabbit polyclonal anti-pSer28/pThr31 antibodies, respectively.

Techniques: Staining, Western Blot

Journal: eLife

Article Title: Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20 Slp1 under stress

doi: 10.7554/eLife.97896

Figure Lengend Snippet: ( A ) In vitro binding assay using bacterially expressed MBP-Slp1 Cdc20 and yeast lysates prepared from nda3-KM311 pmk1-HA-6His cells arrested at 18°C for 6 hr. Note that weak band detected in MBP sample was due to unspecific background binding to amylose beads. Coomassie blue staining shows inputs for MBP and MBP-Slp1 Cdc20 . ( B ) Schematic depiction of the S. pombe Slp1 Cdc20 protein structure with the positions of two confirmed basic-residue patches mediating Slp1 Cdc20 –Pmk1 association indicated by pink bars. Alignment highlights the conservation of basic-residue patches within four Schizosaccharomyces species. The deduced Pmk1-docking motifs and different versions of motif mutations are shown. MIM, Mad2-interaction motif; IR, isoleucine–arginine tail. ( C ) In vitro GST pull-down assays with bacterially expressed recombinant GST-Pmk1 and MBP fusions of wild-type Slp1 Cdc20 or Slp1 Cdc20 mutants harboring Pmk1-docking motif mutations. An aliquot of the same amount of MBP-Slp1 Cdc20 as that added in each GST pull-down reaction was immobilized by amylose resin as the input control. Asterisks indicate unspecific or degraded protein bands. ( D ) Immunoblot analysis of Slp1 Cdc20 abundance in nda3-KM311 cells with indicated genotypes treated at 18°C for 6 hr. Slp1 Cdc20 levels were quantified as in . The experiment was repeated three times. The mean value for each sample was calculated, and p values were calculated against wild-type or pek1 DD cells. ( E ) Time-course analyses of spindle assembly checkpoint (SAC) activation and inactivation in nda3-KM311 cdc13-GFP strains with indicated genotypes. For each time point, ≥300 cells were counted for every sample. The experiment was repeated three times and the mean value for each sample was calculated as in . ( F ) Schematic summarizing the negative effect of Pmk1–Slp1 Cdc20 association on Slp1 Cdc20 abundance and anaphase-promoting complex/cyclosome (APC/C) activation. Figure 3—source data 1. Uncropped blots for . Figure 3—source data 2. Raw data of Slp1 level measurement and time-course analyses of Cdc13-GFP at spindle pole body (SPB) for . Figure 3—source data 3. Raw unedited blots for .

Article Snippet: To remove Slp1 Cdc20 phosphorylation, one of the immunoprecipitated samples was treated with 400 units of Lambda protein phosphatase (New England Biolabs, P0753S) at 30°C for 30 min. Co-immunoprecipitated Slp1 Cdc20 was detected with rabbit polyclonal anti-Slp1 antibodies, and the phosphorylated form of Slp1 Cdc20 was detected with rabbit polyclonal anti-pSer28/pThr31 antibodies, respectively.

Techniques: In Vitro, Binding Assay, Staining, Residue, Recombinant, Control, Western Blot, Activation Assay

Journal: eLife

Article Title: Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20 Slp1 under stress

doi: 10.7554/eLife.97896

Figure Lengend Snippet: ( A ) Schematic depiction of the S. pombe ( Schizosaccharomyces pombe ) Slp1 protein with its homologs in seven other fungi species: Schizosaccharomyces octosporus , Schizosaccharomyces cryophilus , Schizosaccharomyces japonicas , Protomyces lactucae-debilis , Neolecta irregularis , Pneumocystis jirovecii , and Hirsutella rhossiliensis . Positions of amino acids corresponding to seven WD40 repeats and IR (isoleucine–arginine) motif are indicated. ( B ) Local sequence alignment performed with S. pombe Slp1 and its homolog sequences from seven other fungi species. Five potential basic-residue patches within Slp1 and their conserved positions in other species are highlighted in blue. Phosphorylated or ubiquitylated residues detected by mass spectrometry in S. pombe Slp1 together with their corresponding residues in homologs from seven other fungi species are labeled with red frames or red letters, respectively. Lysine 472 (K472) in S. pombe Slp1 and its corresponding residues in homologs from seven other fungi species are also indicated in pink. Note that only aligned local sequences (N-terminal, middle and C-terminal portions) of Slp1 homologs harboring the basic-residue patches are shown.

Article Snippet: To remove Slp1 Cdc20 phosphorylation, one of the immunoprecipitated samples was treated with 400 units of Lambda protein phosphatase (New England Biolabs, P0753S) at 30°C for 30 min. Co-immunoprecipitated Slp1 Cdc20 was detected with rabbit polyclonal anti-Slp1 antibodies, and the phosphorylated form of Slp1 Cdc20 was detected with rabbit polyclonal anti-pSer28/pThr31 antibodies, respectively.

Techniques: Sequencing, Residue, Mass Spectrometry, Labeling

Journal: eLife

Article Title: Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20 Slp1 under stress

doi: 10.7554/eLife.97896

Figure Lengend Snippet: ( A ) Schematic depiction of the S. pombe Slp1 protein with five basic-residue patches potentially required for Slp1–Pmk1 association indicated, and two confirmed interaction-mediating patches highlighted in red. MIM, Mad2-interaction motif; IR, isoleucine–arginine tail. ( B ) In vitro GST pull-down assays were performed with bacterially expressed GST-Pmk1 and MBP-Slp1 with wild-type Slp1 or mutants harboring lysine/arginine (K/R) to glutamic acid (E) mutations of basic-residue patches within Slp1. Proteins bound on GST beads were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and visualized by Coomassie blue staining. Note that only Slp1 harboring clustered mutations K19E, K20E and R21E, or K47E and R48E within two most N-terminal basic-residue patches strongly compromised interaction between Slp1 and Pmk1. Figure 3—figure supplement 2—source data 1. Uncropped gels for . Figure 3—figure supplement 2—source data 2. Full raw unedited Coomassie gel (bead-bound GST-Pmk1 and MBP-Slp1) for . Figure 3—figure supplement 2—source data 3. Full raw unedited Coomassie gel (MBP-Slp1 input) for .

Article Snippet: To remove Slp1 Cdc20 phosphorylation, one of the immunoprecipitated samples was treated with 400 units of Lambda protein phosphatase (New England Biolabs, P0753S) at 30°C for 30 min. Co-immunoprecipitated Slp1 Cdc20 was detected with rabbit polyclonal anti-Slp1 antibodies, and the phosphorylated form of Slp1 Cdc20 was detected with rabbit polyclonal anti-pSer28/pThr31 antibodies, respectively.

Techniques: Residue, In Vitro, Polyacrylamide Gel Electrophoresis, SDS Page, Staining

Journal: eLife

Article Title: Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20 Slp1 under stress

doi: 10.7554/eLife.97896

Figure Lengend Snippet: ( A ) Non-radioactive in vitro phosphorylation assays with bacterially expressed recombinant GST-Slp1 Cdc20 and Pmk1-HA-His purified from yeast cells. The incorporation of the thiophosphate group was determined using anti-thiophosphate ester antibodies (anti-thioP) as indicative of phosphorylation. Note that the presence or absence of GST-Pek1 DD does not affect Slp1 Cdc20 phosphorylation efficiency. A known Pmk1 substrate Atf1 was used as a positive control (Atf1-P). Asterisks indicate bands corresponding to unspecific or likely degraded proteins. ( B ) Summary of mass spectrometry data on Slp1 Cdc20 phosphorylation and ubiquitylation in vivo in nda3-KM311 -arrested cells. Red arrows and filled blue circles denote all detected phosphorylated or ubiquitylated sites, respectively, while gray arrows and unfilled circles indicate the absence of phosphorylation or ubiquitylation in some of these sites, respectively. Alignment highlights the conservation of most of the detected phosphorylation and ubiquitylation sites within four Schizosaccharomyces species. ( C ) In vitro phosphorylation assays with bacterially expressed recombinant GST-fusion of Slp1 Cdc20 fragment (456–488aa), GST-Pmk1 and GST-Pek1 DD . The reactions were blotted with pT480 antibodies. Asterisks indicate bands corresponding to unspecific or likely degraded proteins. ( D ) Immunoblot detection of Slp1 Cdc20 phosphorylation at T480 in vivo. GST-slp1(456–488aa) was purified from mts3-1 cells with indicated genotypes arrested at 36°C for 3.5 hr, and detected with anti-GST and anti-pThr480 antibodies. Note that pThr480 is absent in pmk1∆ cells, and enhanced in pek1 DD cells relative to that in wild-type cells. ( E ) In vitro phosphorylation assays with bacterially expressed recombinant MBP-fusion of Slp1 Cdc20 fragment (1–190aa) and Cdc13 (cyclin B)-containing Cdk1 complexes purified from metaphase-arrested nda3-KM311 yeast cells. 1-NM-PP1 was added as inhibitor for analog-sensitive Cdc2-as. The reactions were blotted with pS28/pT31 antibodies. Asterisks indicate bands corresponding to unspecific or likely degraded proteins. ( F ) Immunoblot detection of Slp1 Cdc20 phosphorylation at S28/T31 in vivo. Apc15-13myc was immunoprecipitated from nda3-KM311 cells treated at 18°C for 6 hr and the samples were blotted with pS28/pT31 antibodies. One IP sample from wild-type background was treated with λ-phosphatase. For cdc2-asM17 cells, 1-NM-PP1 was added to inactivate Cdc2 during culturing. ( G ) Immunoblot analysis of Slp1 Cdc20 abundance in nda3-KM311 cells treated at 18°C for 6 hr. Slp1 Cdc20 levels were quantified as in . The experiment was repeated three times. The mean value for each sample was calculated, and p values were calculated against wild-type or pek1 DD cells. ( H ) Time-course analyses of spindle assembly checkpoint (SAC) activation and inactivation in nda3-KM311 cdc13-GFP strains with indicated genotypes. For each time point, ≥300 cells were counted for every sample. The experiment was repeated three times and the mean value and p value for each sample were calculated as in . ( I ) Schematic summarizing the negative effect of Slp1 Cdc20 phosphorylation by Pmk1 on its abundance and anaphase-promoting complex/cyclosome (APC/C) activation. Figure 4—source data 1. Uncropped blots for . Figure 4—source data 2. Raw data of Slp1 level measurement and time-course analyses of Cdc13-GFP at spindle pole body (SPB) for . Figure 4—source data 3. Raw unedited blots for .

Article Snippet: To remove Slp1 Cdc20 phosphorylation, one of the immunoprecipitated samples was treated with 400 units of Lambda protein phosphatase (New England Biolabs, P0753S) at 30°C for 30 min. Co-immunoprecipitated Slp1 Cdc20 was detected with rabbit polyclonal anti-Slp1 antibodies, and the phosphorylated form of Slp1 Cdc20 was detected with rabbit polyclonal anti-pSer28/pThr31 antibodies, respectively.

Techniques: In Vitro, Recombinant, Purification, Positive Control, Mass Spectrometry, In Vivo, Western Blot, Immunoprecipitation, Activation Assay

Journal: eLife

Article Title: Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20 Slp1 under stress

doi: 10.7554/eLife.97896

Figure Lengend Snippet: ( A, B ) Anaphase-promoting complex/cyclosome (APC/C) subunits were isolated by immunoprecipitation of Apc15-GFP from nda3-KM311 cells arrested at metaphase by being treated at 18°C for 6 hr. Purified proteins were analyzed by SDS–PAGE and mass spectrometry. Coomassie blue-stained protein gels after SDS–PAGE are shown ( A ). APC/C subunits or related proteins identified by Apc15-GFP purifications followed by mass spectrometry are listed, and percentages of peptide sequence coverage for each protein are indicated ( B ). ( C–E ) Slp1 sequences retrieved from three purifications in indicated strains with peptide sequence coverage (green), phosphorylated serine or threonine (red), and ubiquitylated lysine (blue). Sequences not covered after mass spectrometry analysis are in gray. Figure 4—figure supplement 1—source data 1. Uncropped gels for . Figure 4—figure supplement 1—source data 2. Full raw unedited Coomassie gel ( wild-type ) for . Figure 4—figure supplement 1—source data 3. Full raw unedited Coomassie gel ( pmk1Δ , pek1 DD ) for .

Article Snippet: To remove Slp1 Cdc20 phosphorylation, one of the immunoprecipitated samples was treated with 400 units of Lambda protein phosphatase (New England Biolabs, P0753S) at 30°C for 30 min. Co-immunoprecipitated Slp1 Cdc20 was detected with rabbit polyclonal anti-Slp1 antibodies, and the phosphorylated form of Slp1 Cdc20 was detected with rabbit polyclonal anti-pSer28/pThr31 antibodies, respectively.

Techniques: Isolation, Immunoprecipitation, Purification, SDS Page, Mass Spectrometry, Staining, Sequencing

Journal: eLife

Article Title: Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20 Slp1 under stress

doi: 10.7554/eLife.97896

Figure Lengend Snippet: Examples of spectra for five phosphorylation sites (S28, T31, S59, S76, and T480) and one ubiquitylation site (K479) identified in Apc15-GFP-assocaited Slp1 purified from metaphase-arrested P adh11 -pek1 DD cells.

Article Snippet: To remove Slp1 Cdc20 phosphorylation, one of the immunoprecipitated samples was treated with 400 units of Lambda protein phosphatase (New England Biolabs, P0753S) at 30°C for 30 min. Co-immunoprecipitated Slp1 Cdc20 was detected with rabbit polyclonal anti-Slp1 antibodies, and the phosphorylated form of Slp1 Cdc20 was detected with rabbit polyclonal anti-pSer28/pThr31 antibodies, respectively.

Techniques: Purification

Journal: eLife

Article Title: Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20 Slp1 under stress

doi: 10.7554/eLife.97896

Figure Lengend Snippet: ( A ) Schematic depiction of the experiment design for artificially forced tethering of Slp1 Cdc20 to Pmk1 using GBP–GFP system. ( Left ) Structures of the genomically integrated P adh1 -slp1 (1–60aa) -mEGFP-2xNLS-GST-slp1 (456–488aa) ::nat R at ade6 + locus, P adh11 -pmk1-GBP-mCherry::hyg R at lys1 + locus, and P adh11 -6xHA-pek1 DD ::kan R at ura4 + locus. ( Right ) Cartoon depicting the manipulated targeting of mEGFP-2xNLS-GST fusion with Slp1 fragments to GBP-mCherry fusion of Pmk1 mediated by GBP–GFP binding. Note that Thr480 in Slp1 C-terminus could be phosphorylated by either ectopically expressed Pmk1-GBP-mCherry fusion or endogenous Pmk1 activated by Pek1 DD . ( B ) Representative images of mts3-1 cells expressing mEGFP-2xNLS-GST, GBP-mCherry or 6HA fusion proteins driven by promoters P adh1 or P adh11 . Cells were grown to early log phase in liquid yeast extract (YE) at 25°C, and then collected, fixed, DAPI-stained, and visualized by using fluorescence microscopy. DIC, differential interference contrast microscopy. Scale bar, 5 μm. ( C ) Slp1 (1–60aa) -mEGFP-2xNLS-GST-Slp1 (456–488aa) was purified using GST pull-down from mts3-1 cells with indicated genotypes arrested at 36°C for 3.5 hr, and detected with anti-GST and anti-pThr480 antibodies. Note that pThr480 is absent in pmk1∆ cells, while its phosphorylation level was further enhanced in cells when both Pek1 DD and Pmk1-GBP-mCherry were present compared to cells only expressing either fusions. Blots shown are the representative of three independent experiments. pThr480 levels were normalized with ratio between anti-pThr480-recognized and GST bead-bound Slp1 fragment fusion in cells only expressing Slp1 (1–60aa) -mEGFP-2xNLS-GST-Slp1 (456–488aa) set as 1.0. Error bars indicate mean ± standard deviation of three independent experiments. Two-tailed unpaired t -test was used to derive p values against wild-type cells. Figure 4—figure supplement 3—source data 1. Uncropped blots for . Figure 4—figure supplement 3—source data 2. Raw data of quantitative analysis of Slp1 T480 phosphorylation levels for . Figure 4—figure supplement 3—source data 3. Full raw unedited blot (bead-bound, anti-pT480) for . Figure 4—figure supplement 3—source data 4. Full raw unedited blot (bead-bound, anti-GST) for . Figure 4—figure supplement 3—source data 5. Full raw unedited blot (input, anti-GST) for . Figure 4—figure supplement 3—source data 6. Full raw unedited blot (input, Cdc2) for .

Article Snippet: To remove Slp1 Cdc20 phosphorylation, one of the immunoprecipitated samples was treated with 400 units of Lambda protein phosphatase (New England Biolabs, P0753S) at 30°C for 30 min. Co-immunoprecipitated Slp1 Cdc20 was detected with rabbit polyclonal anti-Slp1 antibodies, and the phosphorylated form of Slp1 Cdc20 was detected with rabbit polyclonal anti-pSer28/pThr31 antibodies, respectively.

Techniques: Binding Assay, Expressing, Staining, Fluorescence, Microscopy, Purification, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20 Slp1 under stress

doi: 10.7554/eLife.97896

Figure Lengend Snippet: Five phosphorylation sites (Ser28, Thr31, Ser59, Ser76, and Thr480) in Slp1 identified in this study are indicated in red. Putative Cdk1 or mitogen-activated protein kinase (MAPK) phosphorylation sites in Slp1 homologs (e.g. Ser41, Thr55, Thr59, Thr70, Thr106, Thr157, Ser408, Ser452, and Ser487 in human Cdc20; Ser50, Thr64, Thr68, and Thr79 in frog Cdc20; and Thr7, Thr32, and Ser87 in worm Cdc20) suggested by previous studies are indicated in blue.

Article Snippet: To remove Slp1 Cdc20 phosphorylation, one of the immunoprecipitated samples was treated with 400 units of Lambda protein phosphatase (New England Biolabs, P0753S) at 30°C for 30 min. Co-immunoprecipitated Slp1 Cdc20 was detected with rabbit polyclonal anti-Slp1 antibodies, and the phosphorylated form of Slp1 Cdc20 was detected with rabbit polyclonal anti-pSer28/pThr31 antibodies, respectively.

Techniques:

Journal: eLife

Article Title: Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20 Slp1 under stress

doi: 10.7554/eLife.97896

Figure Lengend Snippet: Immunoblotting of extracts from asynchronously growing nda3-KM311 cells at 30°C expressing wild-type slp1 + or slp1 with the indicated mutations. An extract from nda3-KM311 slp1 + cells synchronized with HU and arrested at metaphase by treatment at 18°C for 6 hr was loaded as a control. Blots shown are the representative of three independent experiments. Slp1 Cdc20 levels were quantified with the relative ratio between Slp1 Cdc20 and Cdc2 in wild-type strain asynchronously grown 30°C set as 1.0. Error bars indicate mean ± standard deviation of three independent experiments. Two-tailed unpaired t -test was used to derive p values against wild-type cells. Figure 4—figure supplement 5—source data 1. Uncropped blots for . Figure 4—figure supplement 5—source data 2. Raw data of Slp1 level measurement for . Figure 4—figure supplement 5—source data 3. Full raw unedited blot (Slp1) for . Figure 4—figure supplement 5—source data 4. Full raw unedited blot (Cdc2) for .

Article Snippet: To remove Slp1 Cdc20 phosphorylation, one of the immunoprecipitated samples was treated with 400 units of Lambda protein phosphatase (New England Biolabs, P0753S) at 30°C for 30 min. Co-immunoprecipitated Slp1 Cdc20 was detected with rabbit polyclonal anti-Slp1 antibodies, and the phosphorylated form of Slp1 Cdc20 was detected with rabbit polyclonal anti-pSer28/pThr31 antibodies, respectively.

Techniques: Western Blot, Expressing, Control, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20 Slp1 under stress

doi: 10.7554/eLife.97896

Figure Lengend Snippet: ( A ) Immunoblot analysis of Slp1 Cdc20 abundance in apc15 + or apc15 ∆ background cells with indicated genotypes after being treated at 18°C for 6 hr. Slp1 Cdc20 levels were quantified as in . The experiment was repeated three times. The mean value for each sample was calculated, and p values were calculated against wild-type or pek1 DD cells. ( B ) Time-course analyses of spindle assembly checkpoint (SAC) activation and inactivation in nda3-KM311 cdc13-GFP strains with indicated genotypes. The experiments were performed and analyzed as in . ( C ) Immunoblot analysis of Slp1 Cdc20 abundance in K472R, K479R, or K472R/K479R mutants. The experiment was repeated three times. The mean value for each sample was calculated, and p values were calculated against wild-type or pek1 DD cells. ( D ) Time-course analyses of SAC activation and inactivation in nda3-KM311 cdc13-GFP strains with K472R, K479R, or K472R/K479R mutations. The experiments were performed as in ( B ). ( E ) Schematic depiction of affinity pull-down assays using TUBE (tandem ubiquitin-binding entity) agarose beads to detect Slp1 Cdc20 ubiquitylation. ( F ) TUBE pull-down assays in mts3-1 strains carrying sfGFP-tagged wild-type or mutants of Slp1 Cdc20 or pmk1Δ or pek1 DD mutations. The TUBE bead-bound samples were blotted with anti-GFP antibodies. Asterisk indicates unspecific bands recognized by anti-GFP antibodies. ( G ) Schematic summarizing the negative effect of the coupling of Pmk1 phosphorylation and K472/K479-mediated ubiquitylation on Slp1 Cdc20 abundance and anaphase-promoting complex/cyclosome (APC/C) activation. Figure 5—source data 1. Uncropped blots for . Figure 5—source data 2. Raw data of Slp1 level measurement and time-course analyses of Cdc13-GFP at spindle pole body (SPB) for . Figure 5—source data 3. Raw blots unedited for .

Article Snippet: To remove Slp1 Cdc20 phosphorylation, one of the immunoprecipitated samples was treated with 400 units of Lambda protein phosphatase (New England Biolabs, P0753S) at 30°C for 30 min. Co-immunoprecipitated Slp1 Cdc20 was detected with rabbit polyclonal anti-Slp1 antibodies, and the phosphorylated form of Slp1 Cdc20 was detected with rabbit polyclonal anti-pSer28/pThr31 antibodies, respectively.

Techniques: Western Blot, Activation Assay, Binding Assay

Journal: eLife

Article Title: Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20 Slp1 under stress

doi: 10.7554/eLife.97896

Figure Lengend Snippet: Immunoblotting of extracts from asynchronously growing nda3-KM311 cells at 30°C expressing wild-type slp1 + or slp1 with the indicated mutations. A strain with apc15 deletion ( apc15Δ ) was included as a control for comparison, which has been previously shown to have stabilized Slp1 in interphase cells . An extract from nda3-KM311 slp1 + cells synchronized with HU and arrested at metaphase by treatment at 18°C for 6 hr was also loaded as a control. Blots shown are the representative of three independent experiments. Slp1 Cdc20 levels were quantified with the relative ratio between Slp1 Cdc20 and Cdc2 in wild-type strain asynchronously grown at 30°C set as 1.0. Error bars indicate mean ± standard deviation of three independent experiments. Two-tailed unpaired t -test was used to derive p values against wild-type cells. Figure 5—figure supplement 2—source data 1. Uncropped blots for . Figure 5—figure supplement 2—source data 2. Raw data of Slp1 level measurement for . Figure 5—figure supplement 2—source data 3. Full raw unedited blot (Slp1) for . Figure 5—figure supplement 2—source data 4. Full raw unedited blot (Cdc2) for .

Article Snippet: To remove Slp1 Cdc20 phosphorylation, one of the immunoprecipitated samples was treated with 400 units of Lambda protein phosphatase (New England Biolabs, P0753S) at 30°C for 30 min. Co-immunoprecipitated Slp1 Cdc20 was detected with rabbit polyclonal anti-Slp1 antibodies, and the phosphorylated form of Slp1 Cdc20 was detected with rabbit polyclonal anti-pSer28/pThr31 antibodies, respectively.

Techniques: Western Blot, Expressing, Control, Comparison, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20 Slp1 under stress

doi: 10.7554/eLife.97896

Figure Lengend Snippet: Sequence alignment performed with C-terminal tails of S. pombe Slp1 and its Cdc20 homolog sequences from human ( H. sapiens ), frog ( X. laevis ), and worm ( C. elegans ). Lysines residues in fission yeast Slp1 (K472 and K479) and human Cdc20 (K485 and K490), which have been confirmed in this study and previous studies ( ; ), respectively, to be responsible for their ubiquitylation and degradation, are indicated in red. Other lysine residues and flanking threonine or serine residues in these sequences are also highlighted in orange or green, respectively.

Article Snippet: To remove Slp1 Cdc20 phosphorylation, one of the immunoprecipitated samples was treated with 400 units of Lambda protein phosphatase (New England Biolabs, P0753S) at 30°C for 30 min. Co-immunoprecipitated Slp1 Cdc20 was detected with rabbit polyclonal anti-Slp1 antibodies, and the phosphorylated form of Slp1 Cdc20 was detected with rabbit polyclonal anti-pSer28/pThr31 antibodies, respectively.

Techniques: Sequencing

Journal: eLife

Article Title: Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20 Slp1 under stress

doi: 10.7554/eLife.97896

Figure Lengend Snippet: Normal-looking four-spore asci obtained after crosses between nda3-KM311 background strains carrying slp1 + lys1::P slp1 -slp1 (K472R) ::hyg R , slp1 + lys1::P slp1 -slp1 (K479R) ::hyg R or slp1 + lys1::P slp1 -slp1 (K472R;K479R) ::hyg R and slp1Δ::ura4 + lys1::P slp1 -slp1 + ::nat R strain were dissected using a micromanipulator. The genotypes of colonies formed from germinated spores were deduced after being replicated on selective plates. Mutant candidates of nda3-KM311 slp1Δ::ura4 + lys1::P slp1 -slp1 (K472R)/(K479R)/(K472R;K479R) ::hyg R are indicated by light blue circles (normal-looking colonies), yellow circles (small colonies), or dashed red circles (spores failing to germinate) on representative plates. Quantitative analyses of synthetic lethality of desired mutants based on dissected four-spore asci showed that simultaneous mutation of K472 and K479 in Slp1 to arginine (i.e. K472R;K479R double mutant) causes strong synthetic growth defects.

Article Snippet: To remove Slp1 Cdc20 phosphorylation, one of the immunoprecipitated samples was treated with 400 units of Lambda protein phosphatase (New England Biolabs, P0753S) at 30°C for 30 min. Co-immunoprecipitated Slp1 Cdc20 was detected with rabbit polyclonal anti-Slp1 antibodies, and the phosphorylated form of Slp1 Cdc20 was detected with rabbit polyclonal anti-pSer28/pThr31 antibodies, respectively.

Techniques: Mutagenesis

Journal: eLife

Article Title: Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20 Slp1 under stress

doi: 10.7554/eLife.97896

Figure Lengend Snippet: Cultures of mts3-1 his5::P adh11 -pek1 DD ::nat R strains carrying sfGFP-tagged wild-type or mutants of Slp1 Cdc20 with mutations in Pmk1-docking motives (DS-5A, PDSP), ubiquitylation sites (K472R; K479R or K472R/K479R), Pmk1 phosphorylation sites (T480A; S76A/T480A), or combined Pmk1 and Cdk1 phosphorylation sites (3A, 4A, or 5A) were first grown at 25°C to mid-log phase and then shifted to 36°C for 3.5 hr to block cells in mitosis prior to harvesting and cell lysis. The strain mts3-1 his5::P adh11 -pek1 DD ::nat R pmk1Δ served as a negative control. Ubiquitinated proteins were pulled down from yeast lysates using tandem ubiquitin-binding entities (TUBEs). The TUBE bead-bound samples were blotted with anti-GFP antibodies. Asterisks indicate unspecific bands recognized by anti-GFP antibodies. Figure 5—figure supplement 5—source data 1. Uncropped blots for . Figure 5—figure supplement 5—source data 2. Full raw unedited blot (bead-bound sfGFP-Slp1, blot 1) for . Figure 5—figure supplement 5—source data 3. Raw unedited blots for .

Article Snippet: To remove Slp1 Cdc20 phosphorylation, one of the immunoprecipitated samples was treated with 400 units of Lambda protein phosphatase (New England Biolabs, P0753S) at 30°C for 30 min. Co-immunoprecipitated Slp1 Cdc20 was detected with rabbit polyclonal anti-Slp1 antibodies, and the phosphorylated form of Slp1 Cdc20 was detected with rabbit polyclonal anti-pSer28/pThr31 antibodies, respectively.

Techniques: Blocking Assay, Lysis, Negative Control, Binding Assay

Journal: eLife

Article Title: Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20 Slp1 under stress

doi: 10.7554/eLife.97896

Figure Lengend Snippet: ( A ) Schematic depiction of the experimental design for treatment with KCl or caspofungin during nda3 -mediated spindle checkpoint activation to activate mitogen-activated protein kinases (MAPKs). Samples were collected at indicated time points for subsequent analyses including immunoblotting, co-immunoprecipitation (co-IP), and time-course analysis on spindle assembly checkpoint (SAC) or anaphase-promoting complex/cyclosome (APC/C) activation. ( B ) Immunoblot analysis of activation of MAPKs and Slp1 Cdc20 protein levels. Samples with or without indicated treatments were blotted with anti-phospho p42/44 and anti-phospho p38 antibodies as indicative of phosphorylated Pmk1 (Pmk1-P) or phosphorylated Sty1 (Sty1-P), respectively. Slp1 Cdc20 levels were detected with anti-Slp1 antibodies and anti-Cdc2 was used as loading control. ( C ) Co-immunoprecipitation analysis of APC/C–mitotic checkpoint complex (MCC) association upon environmental stress. Lid1-TAP was immunoprecipitated from nda3-KM311 -arrested cells and associated Mad2-GFP, Mad3-GFP and Slp1 Cdc20 were detected as in . Note that APC/C–MCC association was disrupted when 0.6 M KCl was present during cell culturing or during immunoprecipitation procedures. ( D ) Time-course analyses of SAC activation and inactivation in nda3-KM311 cdc13-GFP strains with indicated genotypes after arrest at 18°C and KCl or caspofungin treatments. The experiment was repeated three times and the mean value and p value for each sample were calculated as in . ( E ) Time-course analyses of SAC activation and inactivation efficiency in Pmk1-docking- and phosphorylation-deficient slp1 mutants under environmental stresses elicited by 0.6 M KCl or 2 μg/ml caspofungin. Figure 6—source data 1. Uncropped blots for . Figure 6—source data 2. Raw data of time-course analyses of Cdc13-GFP at spindle pole body (SPB) for . Figure 6—source data 3. Raw unedited blots for .

Article Snippet: To remove Slp1 Cdc20 phosphorylation, one of the immunoprecipitated samples was treated with 400 units of Lambda protein phosphatase (New England Biolabs, P0753S) at 30°C for 30 min. Co-immunoprecipitated Slp1 Cdc20 was detected with rabbit polyclonal anti-Slp1 antibodies, and the phosphorylated form of Slp1 Cdc20 was detected with rabbit polyclonal anti-pSer28/pThr31 antibodies, respectively.

Techniques: Activation Assay, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Control, Cell Culture

Journal: eLife

Article Title: Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20 Slp1 under stress

doi: 10.7554/eLife.97896

Figure Lengend Snippet: Yeast strain carrying P nmt1 -mad2::leu1 + was pre-cultured in minimal medium with supplements (EMM5S) and 15 µM thiamine. Cells were then washed in EMM5S to remove thiamine before being grown in EMM5S at 30°C for 18 hr to induce Mad2 overexpression. Wild-type cells grown in yeast extract (YE) were treated with 0.6 M KCl for 60 min as a positive control for Pmk1 activation. Protein samples after SDS–PAGE were blotted with anti-phospho p42/44 antibodies as indicative of phosphorylated Pmk1 (Pmk1-P). Slp1 Cdc20 levels were detected with anti-Slp1 antibodies and anti-PSTAIRE (detecting Cdc2) was used as loading control. Blots shown are the representative of three independent experiments. Figure 6—figure supplement 1—source data 1. Uncropped blots for . Figure 6—figure supplement 1—source data 2. Full raw unedited blot (phosphorylated Pmk1) for . Figure 6—figure supplement 1—source data 3. Full raw unedited blot (Slp1) for . Figure 6—figure supplement 1—source data 4. Full raw unedited blot (Cdc2) for .

Article Snippet: To remove Slp1 Cdc20 phosphorylation, one of the immunoprecipitated samples was treated with 400 units of Lambda protein phosphatase (New England Biolabs, P0753S) at 30°C for 30 min. Co-immunoprecipitated Slp1 Cdc20 was detected with rabbit polyclonal anti-Slp1 antibodies, and the phosphorylated form of Slp1 Cdc20 was detected with rabbit polyclonal anti-pSer28/pThr31 antibodies, respectively.

Techniques: Cell Culture, Over Expression, Positive Control, Activation Assay, SDS Page, Control

Journal: eLife

Article Title: Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20 Slp1 under stress

doi: 10.7554/eLife.97896

Figure Lengend Snippet: nda3-KM311 strains expressing wild-type slp1 + or slp1 with the indicated mutations were first synchronized with HU and then arrested at metaphase by treatment at 18°C for 5 hr. Then, a final concentration of 0.6 M KCl or 2 μg/ml caspofungin was added into cultures and left at 18°C for 1 hr before harvesting. nda3-KM311 slp1 + cells either grown asynchronously at 30°C or synchronized with HU and arrested at metaphase by treatment at 18°C for 6 hr served as controls. Protein samples were subjected to SDS–PAGE and immunoblotted with anti-phospho p42/44 and anti-phospho p38 antibodies as indicative of phosphorylated Pmk1 (Pmk1-P) or phosphorylated Sty1 (Sty1-P), respectively. Slp1Cdc20 levels were detected with anti-Slp1 antibodies and anti-PSTAIRE (detecting Cdc2) was used as loading control. The experiment was repeated three times. Slp1 Cdc20 levels were quantified with the relative ratio between Slp1 Cdc20 and Cdc2 in wild-type strain synchronized and arrested at 18°C for 6 hr set as 1.0. The mean value for each sample was calculated from three independent experiments, and p values were calculated against wild-type cells synchronized and arrested at 18°C for 6 hr. Asterisks indicate unspecific or degraded protein bands. Figure 6—figure supplement 3—source data 1. Uncropped blots for . Figure 6—figure supplement 3—source data 2. Raw data of Slp1 level measurement for . Figure 6—figure supplement 3—source data 3. Raw unedited blots for .

Article Snippet: To remove Slp1 Cdc20 phosphorylation, one of the immunoprecipitated samples was treated with 400 units of Lambda protein phosphatase (New England Biolabs, P0753S) at 30°C for 30 min. Co-immunoprecipitated Slp1 Cdc20 was detected with rabbit polyclonal anti-Slp1 antibodies, and the phosphorylated form of Slp1 Cdc20 was detected with rabbit polyclonal anti-pSer28/pThr31 antibodies, respectively.

Techniques: Expressing, Concentration Assay, SDS Page, Control

Journal: eLife

Article Title: Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20 Slp1 under stress

doi: 10.7554/eLife.97896

Figure Lengend Snippet: ( A ) Schematic depiction of a possible dual mechanism that how activated MAPK signaling pathways are involved in delaying APC/C activation and spindle assembly checkpoint (SAC) inactivation. Upon SAC and MAPK signaling activation, division of labor between the cell integrity pathway (CIP) and the stress-activated pathway (SAP) enables the phosphorylation of Slp1 Cdc20 and unidentified substrate(s) by Pmk1 and Sty1, respectively, which leads to lowered Slp1 Cdc20 level and enhanced mitotic checkpoint complex (MCC) affinity for APC/C. ( B ) Summary of the alteration of Slp1 Cdc20 protein levels and MCC–APC/C association strength under various conditions examined in this study. White wavy line indicates the absence of Apc15.

Article Snippet: To remove Slp1 Cdc20 phosphorylation, one of the immunoprecipitated samples was treated with 400 units of Lambda protein phosphatase (New England Biolabs, P0753S) at 30°C for 30 min. Co-immunoprecipitated Slp1 Cdc20 was detected with rabbit polyclonal anti-Slp1 antibodies, and the phosphorylated form of Slp1 Cdc20 was detected with rabbit polyclonal anti-pSer28/pThr31 antibodies, respectively.

Techniques: Activation Assay

Journal: Frontiers in immunology

Article Title: Identification of Secretory Leukoprotease Inhibitor As an Endogenous Negative Regulator in Allergic Effector Cells.

doi: 10.3389/fimmu.2017.01538

Figure Lengend Snippet: Figure 1 | Secretory leukoprotease inhibitor (SLPI) is expressed in basophils and eosinophils but not in mast cells. (A) A quantitative RT-PCR of Slpi in s the bone marrow (BM)-derived basophils, eosinophils, mast cells, and BM cells. (B) Basophils and eosinophils were sorted from the spleen cells after the depletion of CD4+CD8+B220+ cells using magnetic separator. Eosinophils are also sorted from the peritoneal cavity (PEC). A quantitative RT-PCR of Slpi were shown in the indicated cells. (C) Immunoblotting of SLPI in the indicated cells. The data were normalized to the expression of β-actin and presented relative to the expression in BM cells. (D,E) Fluorescence microscopy and transmission electron microscopy (TEM) images of BM-derived basophils (BMBs) (D) and BM-derived eosinophils (BMEos). (E) from B6 and Slpi−/− mice. Bright field (BF), SLPI (red) DAPI (blue), Diff-Quick staining, alcian blue staining, and TEM images are shown (scale bar: 2 µm). (A,B) Data were normalized to the housekeeping Rps16 (mean ± SD). n = 4. **P < 0.01. (C–E) Data are representative of three independent experiments.

Article Snippet: The cells were blocked with blocking reagent (Toyobo) for 1 h at room temperature, incubated with biotinylated goat anti-mouse SLPI (R&D Systems) in Can get signal solution A (Toyobo) overnight at 4°C, and then incubated with allophycocyanin-labeled streptavidin in Can get signal solution B (Toyobo) for another 1 h at room temperature.

Techniques: Quantitative RT-PCR, Derivative Assay, Western Blot, Expressing, Fluorescence, Microscopy, Transmission Assay, Electron Microscopy, Diff-Quik, Staining

Journal: Frontiers in immunology

Article Title: Identification of Secretory Leukoprotease Inhibitor As an Endogenous Negative Regulator in Allergic Effector Cells.

doi: 10.3389/fimmu.2017.01538

Figure Lengend Snippet: Figure 2 | Enhanced cytokine production and tryptase activity in Slpi−/− bone marrow-derived basophils (BMBs) after IgE stimulation. (A–D) B6 and Slpi−/− BMBs were incubated with TNP-OVA for 12 h at the indicated concentrations 1 h after the administration of 5 µg/ml anti-TNP-IgE. (A) The interleukin (IL)-4, 6, and 13 levels in supernatants were measured by an ELISA. (B) The enzyme activities of tryptase (left) and chymase (right) in supernatants were determined using MeOSuc-AAPV-pNA and N-Suc-AAPF-pNA substrate, respectively. (C) The percentages of β-HEX released after the administration of the indicated stimulators. (D) The histamine and cysteinyl leukotrienes (CysLT) production in supernatants was measured by an ELISA. (E) B6 and Slpi−/− BMBs were stimulated with TNP-OVA (1 ng/ml) at the indicated time, 1 h after the administration of 5 µg/ml anti-TNP-IgE. Representative immunoblots of the indicated proteins are shown. (A–D) Data are shown as the mean ± SEM of three different basophil cultures.

Article Snippet: The cells were blocked with blocking reagent (Toyobo) for 1 h at room temperature, incubated with biotinylated goat anti-mouse SLPI (R&D Systems) in Can get signal solution A (Toyobo) overnight at 4°C, and then incubated with allophycocyanin-labeled streptavidin in Can get signal solution B (Toyobo) for another 1 h at room temperature.

Techniques: Activity Assay, Derivative Assay, Incubation, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Frontiers in immunology

Article Title: Identification of Secretory Leukoprotease Inhibitor As an Endogenous Negative Regulator in Allergic Effector Cells.

doi: 10.3389/fimmu.2017.01538

Figure Lengend Snippet: Figure 3 | The depletion of basophil secretory leukoprotease inhibitor (SLPI) exacerbates IgE-mediated allergic responses. (A) The experimental protocol of IgE-mediated chronic allergic inflammation in 5-fluorouracil (5-FU)-treated Fcer1g−/− mice adaptively transferred with B6 and Slpi −/− DX5+ cells containing basophils from bone marrow (BM) cells. (B) The kinetics of the ear thickness after the antigen challenge are shown. (C) Ear specimens obtained 6 days after the antigen challenge were stained with HE. Data are representative of three separate experiments and are shown as the mean ± SD. n = 4–6. *P < 0.05, **P < 0.01.

Article Snippet: The cells were blocked with blocking reagent (Toyobo) for 1 h at room temperature, incubated with biotinylated goat anti-mouse SLPI (R&D Systems) in Can get signal solution A (Toyobo) overnight at 4°C, and then incubated with allophycocyanin-labeled streptavidin in Can get signal solution B (Toyobo) for another 1 h at room temperature.

Techniques: Staining

Journal: Frontiers in immunology

Article Title: Identification of Secretory Leukoprotease Inhibitor As an Endogenous Negative Regulator in Allergic Effector Cells.

doi: 10.3389/fimmu.2017.01538

Figure Lengend Snippet: Figure 4 | The absence of secretory leukoprotease inhibitor (SLPI) in BM-derived eosinophils (BMEos) increases interleukin (IL)-6 production and invasive activity. (A) I The production of IL-6 by B6 and Slpi−/− BMEos after lipopolysaccharide (LPS) stimulation for 12 h. (B) B6 and Slpi−/− BMEos were incubated with LPS (1 µg/ml) or IL-33 (0.1 µg/ml) for 12 h. BMEos were also incubated with TNP-OVA (1 ng/ml) for 12 h after the administration of 5 µg/ml anti-TNP-IgE. (A,B) IL-4, 6, and 13 levels in the supernatants of cells were measured by an ELISA. (C) The activities of tryptase and chymase prepared according to the methods described in Figure 2B. (D) The amounts of eosinophil peroxidase (EPO) in B6 and Slpi−/− BMEos after the administration of the indicated stimulators. (E) Chemotactic assays of B6 and Slpi−/− eosinophils by LTB4 (50 nM), CCL2 (50 nM), and CCL11 (10 nM). (F) Invasion assays using Matrigel in B6 and Slpi−/− eosinophils upon LPS (1 µg/ml) and CCL11 (10 nM) stimulation. All of the data are shown as the mean ± SEM of three different eosinophil cultures. * P < 0.05.

Article Snippet: The cells were blocked with blocking reagent (Toyobo) for 1 h at room temperature, incubated with biotinylated goat anti-mouse SLPI (R&D Systems) in Can get signal solution A (Toyobo) overnight at 4°C, and then incubated with allophycocyanin-labeled streptavidin in Can get signal solution B (Toyobo) for another 1 h at room temperature.

Techniques: Derivative Assay, Activity Assay, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in immunology

Article Title: Identification of Secretory Leukoprotease Inhibitor As an Endogenous Negative Regulator in Allergic Effector Cells.

doi: 10.3389/fimmu.2017.01538

Figure Lengend Snippet: Figure 6 | Secretory leukoprotease inhibitor (SLPI) interact with the JNK-interacting protein 3 (JIP3) scaffold protein, and negatively regulates Toll-like receptor (TLR) 4-mediated Elk-1 activation. (A) B6 and Slpi−/− bone marrow-derived eosinophils (BMEos) were stimulated with lipopolysaccharide (LPS) (1 µg/ml). Immunoblots of the indicated proteins are shown. The arrows indicate the p54 and P46 isoforms of JNK1. GAPDH was used as loading and internal monitoring controls. (B) The relative intensities of pJNK1/JNK1 and pSer383 Elk-1/Elk-1 in B6 and Slpi−/− BMEos were estimated by densitometric scanning with normalization to GAPDH (means ± SD). n = 3. *P < 0.05. **P < 0.01. (C) JIP3 and SLPI after the precipitation of anti-JIP3 Ab or control mouse IgG1 in B6 and Slpi−/− BMEos. The loading volumes (1/2 and 1/1) are shown. (A,C) Data are representative of three separate experiments.

Article Snippet: The cells were blocked with blocking reagent (Toyobo) for 1 h at room temperature, incubated with biotinylated goat anti-mouse SLPI (R&D Systems) in Can get signal solution A (Toyobo) overnight at 4°C, and then incubated with allophycocyanin-labeled streptavidin in Can get signal solution B (Toyobo) for another 1 h at room temperature.

Techniques: Activation Assay, Derivative Assay, Western Blot, Control

Journal: Frontiers in immunology

Article Title: Identification of Secretory Leukoprotease Inhibitor As an Endogenous Negative Regulator in Allergic Effector Cells.

doi: 10.3389/fimmu.2017.01538

Figure Lengend Snippet: Figure 5 | Secretory leukoprotease inhibitor (SLPI) transcriptionally regulates the metalloproteinase (MMP)-9 expression in BM-derived eosinophils (BMEos). (A) A DNA microarray analysis of Slpi−/− BMEos before and 3 h after lipopolysaccharide (LPS) (1 µg/ml) stimulation. The relative expression to B6 BMEos is shown. (B) A qRT-PCR of Mmp9 in B6 and Slpi−/− BMEos after LPS (1 µg/ml) stimulation. (C) A qRT-PCR of Mmp9 and Slpi in Slpi−/− BMEos transfected with a plasmid carrying the Slpi gene. (D) Immunoblotting of MMP-9 in the indicated cells. (E) Immunoblotting of MMP-9 in B6 and Slpi−/− BMEos after LPS (1 µg/ml) or interleukin (IL)-5 (10 ng/ml) stimulation for 6 h. (B,C) Data were normalized to the housekeeping Rps16 (mean ± SD). n = 4. ** P < 0.01. (D,E) β-actin was used as a control. Data are representative of three separate experiments.

Article Snippet: The cells were blocked with blocking reagent (Toyobo) for 1 h at room temperature, incubated with biotinylated goat anti-mouse SLPI (R&D Systems) in Can get signal solution A (Toyobo) overnight at 4°C, and then incubated with allophycocyanin-labeled streptavidin in Can get signal solution B (Toyobo) for another 1 h at room temperature.

Techniques: Expressing, Derivative Assay, Microarray, Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot, Control

Journal: Frontiers in immunology

Article Title: Identification of Secretory Leukoprotease Inhibitor As an Endogenous Negative Regulator in Allergic Effector Cells.

doi: 10.3389/fimmu.2017.01538

Figure Lengend Snippet: Figure 7 | The disruption of secretory leukoprotease inhibitor (SLPI) augments eosinophil-mediated airway inflammation. (A–C) The house-dust mite (HDM)- induced asthmatic model. Mice were intranasally sensitized with 1 µg of HDM on Day 0 and were challenged 7 days later with exposure to 1 µg of HDM for 5 consecutive days. (A) The percentages of eosinophils (Siglec-F+ Autofluorescence-) among bronchoalveolar lavage fluid (BALF) cells from B6 and Slpi−/− mice at 72 h after the last HDM challenge. (B) The number of eosinophils among the BALF cells on Day 14. (C) Lung sections from specimens obtained on Day 14 were stained with HE. (D,E) Chitin-induced airway inflammation using an eosinophil adaptive transfer system. CD45.2+ donor B6 or Slpi−/− BMEos were intravenously transferred into allergen-challenged CD45.1+ recipients. (D) The left panel shows the population of CD45.1+ recipient and CD45.2+ donor cells among BALF cells. The right panel shows donor Siglec-F+ F4/80+ eosinophils in CD45.1 recipient mice 1 day after the antigen challenge. (E) The numbers of total cells, recipient eosinophils (CD45.1+CD45.2− Siglec-F+ F4/80+cells), and donor eosinophils among BALF cells are shown. All data are representative of three separate experiments and are shown as the mean ± SD (A,B) n = 8–11, (D,E) n = 4–6. *P < 0.05, **P < 0.01.

Article Snippet: The cells were blocked with blocking reagent (Toyobo) for 1 h at room temperature, incubated with biotinylated goat anti-mouse SLPI (R&D Systems) in Can get signal solution A (Toyobo) overnight at 4°C, and then incubated with allophycocyanin-labeled streptavidin in Can get signal solution B (Toyobo) for another 1 h at room temperature.

Techniques: Disruption, Staining

Journal: American Journal of Translational Research

Article Title: STOML2 as a novel prognostic biomarker modulates cell proliferation, motility and chemo-sensitivity via IL6-Stat3 pathway in head and neck squamous cell carcinoma

doi:

Figure Lengend Snippet: STOML2 expression level is increased in HNSCC and indicates poor prognosis. (A) The frequency of STOML2 somatic alteration across various cancers including HNSCC. (B) The analysis of GEO database (GSE25099 and GSE37991) indicated that the mRNA level of STOML2 was enhanced in HNSCC tissues. N, adjacent normal tissues, C, HNSCC tissues. (C) Representative photographs of IHC results of STOML2 at the invasive margin and the invasive front of HNSCC tissues. (D) Representative photographs of IHC results of STOML2 in HNSCC with different stages of differentiation. Scale bar in (C and D), 100 μm. (E) Kaplan-Meier survival curve showed that HNSCC patients (n=91) with high STOML2 expression level had an unfavorable overall survival (P=0.0379).

Article Snippet: After antigen retrieval, the samples were blocked and incubated with primary antibody against STOML2 (1:200, Proteintech, Rosemont, USA) at 4°C overnight.

Techniques: Expressing

Journal: American Journal of Translational Research

Article Title: STOML2 as a novel prognostic biomarker modulates cell proliferation, motility and chemo-sensitivity via IL6-Stat3 pathway in head and neck squamous cell carcinoma

doi:

Figure Lengend Snippet: STOML2 expression and clinicopathological features of HNSCC

Article Snippet: After antigen retrieval, the samples were blocked and incubated with primary antibody against STOML2 (1:200, Proteintech, Rosemont, USA) at 4°C overnight.

Techniques: Expressing

Journal: American Journal of Translational Research

Article Title: STOML2 as a novel prognostic biomarker modulates cell proliferation, motility and chemo-sensitivity via IL6-Stat3 pathway in head and neck squamous cell carcinoma

doi:

Figure Lengend Snippet: STOML2 expression in HNSCC cell lines. A. The mRNA level of STOML2 was measured in a panel of HNSCC cells by real-time PCR. B. The protein expression of STOML2 was detected in a panel of HNSCC cells by immunoblots. C. Three distinct siRNAs were introduced into both SCC25 and SCC15 cells, respectively. The STOML2 expressions in these cells were measured via real-time PCR and western blotting. Data, mean ± SD, **P<0.01.

Article Snippet: After antigen retrieval, the samples were blocked and incubated with primary antibody against STOML2 (1:200, Proteintech, Rosemont, USA) at 4°C overnight.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: American Journal of Translational Research

Article Title: STOML2 as a novel prognostic biomarker modulates cell proliferation, motility and chemo-sensitivity via IL6-Stat3 pathway in head and neck squamous cell carcinoma

doi:

Figure Lengend Snippet: STOML2 knockdown suppresses proliferation of HNSCC cells. A. The expression of STOML2 in SCC25 and SCC15 cells transfected with a pool of siRNAs against STOML2. B. Growth curve suggested that reduced STOML2 significantly inhibited cell proliferation. Data, mean ± SD, *P<0.05. C. Reduction of colony formation capacity in STOML2-silenced SCC25 and SCC15 cells. Data, mean ± SD, *P<0.05. D. STOML2 knockdown arrested cell cycle at S phase in SCC25 and SCC15 cells.

Article Snippet: After antigen retrieval, the samples were blocked and incubated with primary antibody against STOML2 (1:200, Proteintech, Rosemont, USA) at 4°C overnight.

Techniques: Knockdown, Expressing, Transfection

Journal: American Journal of Translational Research

Article Title: STOML2 as a novel prognostic biomarker modulates cell proliferation, motility and chemo-sensitivity via IL6-Stat3 pathway in head and neck squamous cell carcinoma

doi:

Figure Lengend Snippet: STOML2 knockdown inhibits motility of HNSCC cells in vitro. A. Representative photographs of gap at 0 hr and 24 hr of wound healing assay performed in HNSCC cells transfected with siRNAs against STOML2 or negative control. B. Transwell assay showed that depletion of STOML2 dramatically attenuated the abilities of migration and invasion of SCC25 and SCC15 cells. Scale bar, 100 μm. Data, mean ± SD, *P<0.05, **P<0.01. C. GEO database revealed that MMP9 correlated positively with STOML2. D. Western blotting results showed that STOML2 depletion reduced expression of MMP9.

Article Snippet: After antigen retrieval, the samples were blocked and incubated with primary antibody against STOML2 (1:200, Proteintech, Rosemont, USA) at 4°C overnight.

Techniques: Knockdown, In Vitro, Wound Healing Assay, Transfection, Negative Control, Transwell Assay, Migration, Western Blot, Expressing

Journal: American Journal of Translational Research

Article Title: STOML2 as a novel prognostic biomarker modulates cell proliferation, motility and chemo-sensitivity via IL6-Stat3 pathway in head and neck squamous cell carcinoma

doi:

Figure Lengend Snippet: Reduced STOML2 enhances sensitivity of HNSCC cells to cisplatin in vitro. (A) STOML2 deletion decreased cisplatin IC50 in SCC25 (from 17.08 to 4.161 μM) and SCC15 cells (from 15.12 to 3.847 μM). (B and C) Silenced STOML2 sensitized HNSCC cells to cisplatin treatment and increased apoptosis using flow cytometry (B) and colony formation assay (C). CDDP, cisplatin. Data, mean ± SD, *P<0.05, **P<0.01. (D) Depletion of STOML2 enhanced the level of cleaved Caspase 3 in the case of cisplatin dosing.

Article Snippet: After antigen retrieval, the samples were blocked and incubated with primary antibody against STOML2 (1:200, Proteintech, Rosemont, USA) at 4°C overnight.

Techniques: In Vitro, Flow Cytometry, Colony Assay

Journal: American Journal of Translational Research

Article Title: STOML2 as a novel prognostic biomarker modulates cell proliferation, motility and chemo-sensitivity via IL6-Stat3 pathway in head and neck squamous cell carcinoma

doi:

Figure Lengend Snippet: STOML2 regulates IL6-Stat3 pathway in HNSCC cells. (A) WB results showed that knockdown of STOML2 obviously reduced p-Stat3 (Y705), but affected little on the level of p-Stat3 (S727). (B) Immunofluorescence staining indicated that STOML2 knockdown impeded the accumulation of nuclear p-Stat3. (C) The expression of STOML2 was assessed by western blotting after blocking Stat3 expression. (D) Real-time PCR analysis indicated that STOML2 depletion decreased IL6 mRNA expression. Data, mean ± SD, *P<0.05. (E and F) Western blotting assay revealed that IL6 induced the phosphorylation of Stat3 at Y705 residue (E) and STOML2 regulated the activation of Stat3 in an IL-6 dependent manner (F).

Article Snippet: After antigen retrieval, the samples were blocked and incubated with primary antibody against STOML2 (1:200, Proteintech, Rosemont, USA) at 4°C overnight.

Techniques: Knockdown, Immunofluorescence, Staining, Expressing, Western Blot, Blocking Assay, Real-time Polymerase Chain Reaction, Phospho-proteomics, Residue, Activation Assay

Journal: Journal of medicinal chemistry

Article Title: Discovery of BAY-405: An Azaindole-Based MAP4K1 Inhibitor for the Enhancement of T-Cell Immunity against Cancer.

doi: 10.1021/acs.jmedchem.4c01325

Figure Lengend Snippet: Figure 4. In vitro Pharmacology of BAY-405. (A) Inhibition of MAP4K1 activity in Jurkat T-cells by BAY-405 as measured on the basis of the intracellular levels of phospho-SER376-SLP76 (pSLP76), using MAP4K1 knockdown Jurkat T-cells as controls (Supporting Information Figure S2A). Jurkat T-cells were stimulated with plate-bound anti-CD3 Abs (1 mg/mL) for 30 min after which the pSLP76 levels were determined by a HTRF-based method. (B) Dose-dependent enhancement of T-cell reactivity by BAY-405 and its predecessor BAY-755 in human PBMC cultures stimulated with 30 ng/mL anti-CD3 Ab in the presence of 1 μM PGE2. Secreted IFNγ was analyzed after 24 h by means of ELISA. 2500 nM BAY- 405 vs vehicle p = 0.0024; 156 nM BAY-755 vs vehicle p = 0.0059 (student t-test). (C) T-cell reactivity assay as in (B), but performed in the presence of 20 ng/mL human TGFβ. 2500 nM BAY-405 vs vehicle p = 0.0012; 156 nM BAY-755 vs vehicle p = 0.0028 (student t-test). (D,E) T- cell assays as in (A,B) showing the impact of 1 μM BAY-405 on T-cell activation in the presence of different concentrations of anti-CD3 Ab as well as in the presence and absence of 1 μM PGE2 or 20 ng/mL human TGFβ. PGE2 + BAY-405 vs PGE2 + vehicle p < 0.0001. BAY-405 vs vehicle p = 0.7175; TGFβ + BAY-405 vs TGFβ + vehicle p < 0.0001. BAY-405 vs vehicle p = 0.2195 (ANOVA). (F) T-cell reactivity assay with mouse splenocytes from wild-type and MAP4K1 kinase dead knock in mice (Supporting Information Figure S2B,C) in the presence of 300 ng/mL anti- CD3 Ab and 1 μM PGE2 as well as 500 nM BAY-405 or vehicle. WT vehicle vs KI vehicle p = 0.002; WT vehicle vs WT BAY-405 p = 0.0108; KI vehicle vs KI BAY-405 p = 0.4899 (Student’s t-test).

Article Snippet: Respective membranes were processed and separately incubated with primary antibodies proprietary antimouse pSer376-SLP76 antibody ASH-1−13−5 (rabbit mAb, custom-made by Abcam), SLP76 (Cell Signaling #4958), MAP4K1 (HPK1, Cell Signaling #4472), and GAPDH (Zytomed #RGM2− 6C5).

Techniques: In Vitro, Inhibition, Activity Assay, Knockdown, Enzyme-linked Immunosorbent Assay, Activation Assay, Knock-In

Journal: Journal of medicinal chemistry

Article Title: Discovery of BAY-405: An Azaindole-Based MAP4K1 Inhibitor for the Enhancement of T-Cell Immunity against Cancer.

doi: 10.1021/acs.jmedchem.4c01325

Figure Lengend Snippet: Figure 5. Enhancement of in vitro and in vivo antitumor T-cell reactivity. (A) xCelligence real time analysis of the cytotoxicity of MART-1-specific human T-cells toward the HLA-A*0201, MART-1 positive human melanoma cell line COLO800 in the presence of indicated concentrations of BAY-405. P-value represents the statistical differences at the 100 h time point (Student’s t-test). (B) Systemic exposure of BAY-405 (unbound plasma concentration) after a single oral dose in a mouse as indicated. Indicated in the graph are the BAY-405 IC50 levels determined in the relevant biochemical and cellular assays. See Supporting Information Table S9 for underlying data. (C) Impact of indicated b.i.d. doses of BAY-405 on tumor outgrowth in lungs after i.v. injection of 2 × 104 B16-OVA cells. The numbers of lung nodes were analyzed at day 14 after tumor cell injection. Nine mice were used under each condition. Vehicle vs 30 mg/kg p = 0.0219. Vehicle vs 60 mg/kg p = 0.0001 (Student’s t-test). The experiment was repeated twice with similar outcomes (See Figure 5G and Supporting Information Figure S4B). (D) Suppression of tumor outgrowth by indicated doses of BAY-405 in mice subcutaneously challenged with 2 × 10 × 105 of B16-OVA cells. Four million OT-I were injected iv at day 7. Treatment with BAY-405 started at day 8 and lasted until day 18. Nine mice were used for each condition. Vehicle vs 60 mg/kg b.i.d. p = 0.0393 (ANOVA). (E) Suppression of tumor outgrowth by indicated doses of BAY-405 in mice subcutaneously challenged with 5 × 105 of EMT6 tumor cells. Ten mice were used for each condition. Vehicle vs 60 mg/kg b.i.d. p = 0.0026; vehicle vs 100 mg/kg b.i.d. p = 0.0002 (ANOVA). (F) Suppression pf pSLP76 levels in splenocytes of mice treated with BAY-405 at 60 mg/kg, 100 mg/kg or vehicle b.i.d., as detected by Immunoblotting using a proprietary antimouse pSer376-SLP76 antibody. From 8 mice of each treatment group, spleen samples were taken 1 h after the last compound treatment reflecting Cmax. P-value: vehicle vs BAY-405 60 mg/kg = 0.0006; vehicle vs BAY-405 100 mg/kg > 0.0001. (G) Impact of 60 mg/kg b.i.d. BAY-405, conc. PD-L1 blocking Ab (10 mg/kg twice weekly) and/or a combination thereof on B16-OVA melanoma tumor outgrowth in lungs after i.v. injection of 20,000 tumor cells. The numbers of lung tumor nodes were analyzed at day 14 after tumor grafting. Nine mice were used for each condition. Vehicle vs BAY-405 p = 0.0002; vehicle vs PD-L1 Ab p = 0.0472; vehicle vs BAY-405/PD-L1 Ab p < 0.0001; PD-L1 Ab vs BAY-405/PD-L1 Ab p < 0.0001 (Student’s t-test). (H) Suppression of B16 melanoma outgrowth by 60 mg/kg b.i.d. BAY- 405, 10 mg/kg PD-L1 blocking Ab (twice weekly) and/or a combination thereof in mice subcutaneously challenged with 200,000 B16-OVA melanoma cells; 5 × 106 splenocytes from OT-I mice were injected i.v. at day 6 and 11. Treatment with BAY-405 started at day 7 and lasted until day 17. Ten mice were used for each condition. Vehicle vs BAY-405 p = 0.0064; vehicle vs PD-L1 Ab p = 0.0045; PD-L1 Ab vs BAY-405/PD-L1 Ab p < 0.0146 (ANOVA). Experiment was repeated twice.

Article Snippet: Respective membranes were processed and separately incubated with primary antibodies proprietary antimouse pSer376-SLP76 antibody ASH-1−13−5 (rabbit mAb, custom-made by Abcam), SLP76 (Cell Signaling #4958), MAP4K1 (HPK1, Cell Signaling #4472), and GAPDH (Zytomed #RGM2− 6C5).

Techniques: In Vitro, In Vivo, Clinical Proteomics, Concentration Assay, Injection, Western Blot, Blocking Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Potent antimycobacterial activity of mouse secretory leukocyte protease inhibitor.

doi: 10.4049/jimmunol.180.6.4032

Figure Lengend Snippet: FIGURE 1. Expression of SLPI during mycobacterium infection. A, Wild-type mice were intratracheally infected with BCG (4 105 CFU). At the indicated periods, total RNA was extracted from the lungs. SLPI mRNA expression was analyzed by quantitative real-time RT-PCR. Data are shown as the relative mRNA levels normalized by the corresponding 18S rRNA level. B and C, At 2 days after intratracheal infection with BCG, lung tissue sections were stained with an anti-SLPI Ab (red) and 4,6-diamidino-2-phenylindole (blue) and visualized by fluorescence microscopy. D, BALF was collected at the indicated periods after BCG infection. Mouse SLPI protein expression was analyzed by Western blotting with an anti-SLPI Ab. Data obtained from two independent mice (0, 1, and 2 days) are indicated. E, AEC were incubated with the same number of BCG for the indicated periods. SLPI mRNA expression was analyzed by quantitative real-time RT-PCR. Data are shown as the relative mRNA levels normalized by the corresponding 18S rRNA level. F, AEC were incubated with the same number of BCG. Culture supernatants were collected before () and after 24 h of infection () and subjected to Western blot analysis using an anti-SLPI Ab. Data obtained from two independent cell clones are shown. G, Alveolar macrophages were collected from uninfected wild-type mice, cultured with or without BCG for 48 h, and then analyzed for their SLPI mRNA expression by quantitative real-time RT-PCR. The results are presented as the mean SD.

Article Snippet: After blocking with 5% milk, the membrane was incubated with the above-described biotinylated anti-mouse SLPI Ab (0.2 g/ml) and a streptavidin-HRP complex (1/10,000 dilution; R&D Systems).

Techniques: Expressing, Infection, Quantitative RT-PCR, Staining, Microscopy, Western Blot, Incubation, Clone Assay, Cell Culture

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Potent antimycobacterial activity of mouse secretory leukocyte protease inhibitor.

doi: 10.4049/jimmunol.180.6.4032

Figure Lengend Snippet: FIGURE 3. SLPI associates with BCG. SLPI and BSA were labeled with FLUOS (Roche). Labeled proteins were incubated with BCG for 30 min, and analyzed by fluorescence microscopy.

Article Snippet: After blocking with 5% milk, the membrane was incubated with the above-described biotinylated anti-mouse SLPI Ab (0.2 g/ml) and a streptavidin-HRP complex (1/10,000 dilution; R&D Systems).

Techniques: Labeling, Incubation, Microscopy

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Potent antimycobacterial activity of mouse secretory leukocyte protease inhibitor.

doi: 10.4049/jimmunol.180.6.4032

Figure Lengend Snippet: FIGURE 4. SLPI disrupts the BCG cell membrane. A, BCG was incubated with or without SLPI for the indicated periods and observed with scanning electron microscopy. B, The indicated concentrations of SLPI were added to a BCG suspension containing NPN, and the NPN fluorescence was moni- tored for the indicated periods. Repre- sentative data of three independent ex- periments are shown.

Article Snippet: After blocking with 5% milk, the membrane was incubated with the above-described biotinylated anti-mouse SLPI Ab (0.2 g/ml) and a streptavidin-HRP complex (1/10,000 dilution; R&D Systems).

Techniques: Membrane, Incubation, Electron Microscopy, Suspension

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Potent antimycobacterial activity of mouse secretory leukocyte protease inhibitor.

doi: 10.4049/jimmunol.180.6.4032

Figure Lengend Snippet: FIGURE 7. Slpi/ mice are highly susceptible to M. tuberculosis infection. A, M. tuberculosis (4 105 CFU) were intratracheally infected into wild-type and Slpi/ mice and their survival was monitored. B, M. tuberculosis (4 105 CFU) were intratracheally infected into wild-type and Slpi/

Article Snippet: After blocking with 5% milk, the membrane was incubated with the above-described biotinylated anti-mouse SLPI Ab (0.2 g/ml) and a streptavidin-HRP complex (1/10,000 dilution; R&D Systems).

Techniques: Infection

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: SLP2 is identified as an interacting partner of MIC13 (A) Interactome of MIC13 with co-IP (co-immunoprecipitation) coupled mass spectrometry revealed SLP2 as an interactor of MIC13. (B) The interaction between SLP2 and MIC13 was confirmed by co-IP using FLAG antibody in isolated mitochondria from MIC13 KO cells stably expressing MIC13-FLAG or empty vector (EV) pMSCVpuro as background control. I: input lanes represent loading of 10% of total lysates, E: eluate represent proteins eluted from anti-Flag M2 beads, ∗ non-specific IgG bands. (C) Co-IP was used to detect the SLP2-MICOS interaction using isolated mitochondria from SLP2 KO stably expressing pMSCVpuro EV (background control) or SLP2-MYC. Co-IP was performed using MYC-Trap agarose beads. I: Input fraction (10% of total lysate), E: Eluate fraction. YME1L was used as a positive interactor of SLP2 whereas Mt-CO2 and HSP60 served as non-interactors. All the MICOS subunits were detected in the elution fraction from SLP2-MYC co-IP. (D) Proximity ligation assay (PLA) in HeLa cells with antibodies against MICOS subunits and SLP2. PLA signals are shown as red spots indicating respective protein interactions. SLP2 alone and Mt-CO2 & SLP2 antibodies were probed as negative controls. Scale bar 20 μm. (E) BN-PAGE with isolated mitochondria from WT cells revealed a co-migration pattern of SLP2 with higher molecular weight MICOS complex. (F) A heatmap and graph represent the normalized occurrence of SLP2 and MICOS subunits in complexome profiling data obtained from HEK293 cells studied previously. SLP2 co-clustered with high molecular weight MICOS complex at around 2000 kDa. (G) Scaffolding model depicting interaction of SLP2 with MICOS subunits shows that SLP2 provides a scaffold for interaction of MICOS subunits.

Article Snippet: Blocking solution was removed and primary antibodies with 1:100 dilution ratio was added to the samples and incubation was carried out at 37°C for 2 h. The following primary antibodies were used: MIC10 (Abcam, 84969), MIC13 (custom made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625-1-AP), MIC25 (Proteintech, 20639-1-AP), MIC26 (Thermofisher Scientific, MA5-15493), MIC27 (Sigma-Aldrich, HPA000612-100UL), MIC60 (Abcam, ab110329), SLP2 (Abcam, ab102051), SLP2 (OriGene, TA808240), Mt-CO2 (Abcam, ab110258)), OPA1 (custom-made, Pineda against human OPA1 using peptide CDLKKVREIQEKLDAFIEALHQEK, Vinculin (Sigma-Aldrich, V9131).

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Mass Spectrometry, Isolation, Stable Transfection, Expressing, Plasmid Preparation, Control, Proximity Ligation Assay, Migration, Molecular Weight, Scaffolding

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: Loss of SLP2 leads to aberrant cristae structure and reduced MIC26 levels (A) Steady state levels of MICOS proteins with western blot analysis from WT, SLP2 KO and SLP2 KO cells stably expressing pMSCVpuro EV or SLP2-MYC. Tubulin serves as a loading control for western blots. (B) Western blot quantification depicting relative protein levels. SLP2 KO was normalized to WT and SLP2 KO + SLP2 MYC was normalized to SLP2 KO + EV samples. Data is represented as mean ± standard error of mean ( n = 3). Statistical analysis was performed using Student’s t test. ∗ p -value ≤0.05, ∗∗∗ p -value ≤0.001. MIC26 levels were reduced in SLP2 KO. Notably, MIC27 levels showed a slight increase in SLP2 KO. (C) Western blot analysis of steady state levels of MICOS proteins from WT and SLP2 KO cells stably expressing pGIPZ-control shRNA or YME1L shRNA. The results are depicted by a model illustrating the role of SLP2 in stabilizing MIC26 through the regulation of YME1L-mediated proteolysis. HSP60 acts as an internal protein loading control. (D) TEM images from WT, SLP2 KO and MIC26 KO cells. SLP2 KO shows loss of cristae and CJs with accumulation of swollen cristae, while MIC26 KO shows slight cristae branching. Scale bar represents 500 nm. The skeletonization of the TEM image is depicted on right side. (E) Cristae number and CJs per mitochondrial section quantified from TEM images. Statistical analysis was performed using Student’s t test. ∗ p -value ≤0.05, ∗∗ p -value ≤0.01, ∗∗∗∗ p -value ≤0.0001, ns = non-significant, p -value >0.05. (F) Assessment of mitochondrial morphologies from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells untreated or post treatment with 10 μM cycloheximide for 2 h. Scale bar represented as 15 μm. (G) Percentage of cells displaying tubular, intermediate, fragmented or hyperfused mitochondria ( n = 3). Statistical analysis was performed using Student’s t test. ∗∗ p -value ≤0.01, ∗∗∗ p -value ≤0.001. Data represented as mean ± standard error of mean.

Article Snippet: Blocking solution was removed and primary antibodies with 1:100 dilution ratio was added to the samples and incubation was carried out at 37°C for 2 h. The following primary antibodies were used: MIC10 (Abcam, 84969), MIC13 (custom made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625-1-AP), MIC25 (Proteintech, 20639-1-AP), MIC26 (Thermofisher Scientific, MA5-15493), MIC27 (Sigma-Aldrich, HPA000612-100UL), MIC60 (Abcam, ab110329), SLP2 (Abcam, ab102051), SLP2 (OriGene, TA808240), Mt-CO2 (Abcam, ab110258)), OPA1 (custom-made, Pineda against human OPA1 using peptide CDLKKVREIQEKLDAFIEALHQEK, Vinculin (Sigma-Aldrich, V9131).

Techniques: Western Blot, Stable Transfection, Expressing, Control, shRNA

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: SLP2 and MIC13 synergistically modulate assembly and nanoscale distribution of MIC60 (A) Assessment of steady state levels of MICOS proteins with western blot from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells. HSP60 serves as a loading control. (B) BN-PAGE of isolated mitochondria from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells to assess MICOS assembly. MIC13-SLP2 DKO showed reduced MIC60 assembly in MICOS complex compared to any single KO. OXPHOS cocktail antibody is used as a control in BN-PAGE. (C) BN-PAGE quantification depicting relative protein assembly levels normalized to Coomassie. Normalized intensities of MIC60 antibody signal throughout gel were used to calculate fold change relative to WT. Data is represented as mean ± standard error of mean, MIC60 ( n = 5), MIC10 ( n = 4), OXPHOS ( n = 3). Statistical analysis was performed using Student’s t test. ∗ p -value ≤0.05, ∗∗ p -value ≤0.01, ∗∗∗ p -value ≤0.001, ∗∗∗∗ p -value ≤0.0001, ns = non-significant, p -value >0.05. (D) STED nanoscopy images from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells displaying MIC60 punctae. Images on the left side show individual mitochondria that are delineated by dotted lines. Images on the right-side zooms into the boxed mitochondria region. Blue arrows indicate individual MIC60 punctae in a rail-like arrangement in WT cells. Green arrow represents perturbed MIC60 puncta in MIC13 KO and SLP2 KO that are not arranged in rail-like arrangement. Purple arrow shows evenly spread (diffuse) MIC60 puncta in MIC13-SLP2 DKO cells suggesting an altered pattern compared to WT. Scale bars represent the 500 nm. (E) Percentage of individual mitochondria displaying MIC60 puncta predominately arranged as rail-like pattern, non-rail-like pattern, or diffuse pattern is presented in a pie-chart format. (F) A model depicting that MIC60-subcomplex and MIB assembly is dependent on SLP2-MIC13.

Article Snippet: Blocking solution was removed and primary antibodies with 1:100 dilution ratio was added to the samples and incubation was carried out at 37°C for 2 h. The following primary antibodies were used: MIC10 (Abcam, 84969), MIC13 (custom made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625-1-AP), MIC25 (Proteintech, 20639-1-AP), MIC26 (Thermofisher Scientific, MA5-15493), MIC27 (Sigma-Aldrich, HPA000612-100UL), MIC60 (Abcam, ab110329), SLP2 (Abcam, ab102051), SLP2 (OriGene, TA808240), Mt-CO2 (Abcam, ab110258)), OPA1 (custom-made, Pineda against human OPA1 using peptide CDLKKVREIQEKLDAFIEALHQEK, Vinculin (Sigma-Aldrich, V9131).

Techniques: Western Blot, Control, Isolation

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: SLP2 specifically regulates assembly kinetics of MIC60 (A) WT cells stably expressing pLIX403 EV and MIC13 KO, MIC13-SLP2 DKO cells stably expressing pLIX403-MIC13-FLAG were treated with 1 μg/mL of doxycycline (Dox) for indicated time points and western blot analysis depicting steady state levels of MICOS proteins upon induction of MIC13-FLAG are shown. HSP60 serves as a loading control. (B) BN-PAGE with isolated mitochondria from WT cells stably expressing pLIX403 EV, and MIC13 KO and MIC13-SLP2 DKO cells stably expressing pLIX403-MIC13-FLAG treated with 1 μg/mL of doxycycline (Dox) for indicated time points showing stable incorporation of MIC13-FLAG in MICOS complex. (C) BN-PAGE with isolated mitochondria from WT cells stably expressing pLIX403 EV, and MIC13 KO and MIC13-SLP2 DKO cells stably expressing pLIX403-MIC13-FLAG treated with 1 μg/mL of Dox for indicated time points was probed for MIC10, MIC27 and MIC60 antibody. The green arrow (in the MIC13 KO lane) and red arrow (in the MIC13-SLP2 DKO lane) highlight the delay in assembly of MIC60 at the 8-h timepoint. The assembly kinetics of the MIC60-subcomplex, rather than the MIC10-subcomplex, is dependent on SLP2. (D) BN-PAGE quantification depicting relative protein assembly levels of MIC60 at 8 h normalized to Coomassie. Normalized intensities of MIC60 antibody signal throughout gel were used to calculate fold change relative to WT. Data is represented as mean ± standard error of mean ( n = 3). The quantification reveals delayed MIC60 assembly in the absence of SLP2. Statistical analysis was performed using Student’s t test. ∗ p -value ≤0.05. (E) A model depicting the assembly kinetics of MIC60 in MICOS complex depends on SLP2. Slower incorporation of MIC60 is observed in the absence of SLP2 upon reintroduction of MIC13 in MIC13-SLP2 DKO .

Article Snippet: Blocking solution was removed and primary antibodies with 1:100 dilution ratio was added to the samples and incubation was carried out at 37°C for 2 h. The following primary antibodies were used: MIC10 (Abcam, 84969), MIC13 (custom made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625-1-AP), MIC25 (Proteintech, 20639-1-AP), MIC26 (Thermofisher Scientific, MA5-15493), MIC27 (Sigma-Aldrich, HPA000612-100UL), MIC60 (Abcam, ab110329), SLP2 (Abcam, ab102051), SLP2 (OriGene, TA808240), Mt-CO2 (Abcam, ab110258)), OPA1 (custom-made, Pineda against human OPA1 using peptide CDLKKVREIQEKLDAFIEALHQEK, Vinculin (Sigma-Aldrich, V9131).

Techniques: Stable Transfection, Expressing, Western Blot, Control, Isolation

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: Stabilized MIC10-subcomplex for MIC60 seeding and CJ formation (A) Western blot analysis of WT, MIC13 KO, SLP2 KO, MIC13-SLP2 DKO stably expressing pGIPZ-Control shRNA or pGIPZ-YME1L shRNA (KD) to assess steady state levels of MICOS proteins. The steady state levels of MIC60 remain unaltered across different cell lines. HSP60 serves as a loading control. (B) BN-PAGE with isolated mitochondria from WT, MIC13 KO, SLP2 KO, MIC13-SLP2 DKO stably expressing pGIPZ-Control shRNA or pGIPZ- YME1L shRNA. The stabilized MIC10-subcomplex upon YME1L depletion could partially rescue the incorporation of MIC60 and MTX assembly in MIC13-SLP2 DKO. This shows that MIC10-subcomplex provides a docking site for assembly of MIC60. (C) BN-PAGE quantification depicting relative protein assembly levels normalized to Coomassie. Normalized intensities of MIC60 antibody signal throughout gel were used to calculate fold change relative to WT. Data is represented as mean ± standard error of mean ( n = 3). Statistical analysis was performed using Student’s t test. ∗ p -value ≤0.05, ∗∗ p -value ≤0.01, ∗∗∗ p -value ≤0.001, ns = non-significant, p -value >0.05. (D) Mitochondrial cristae morphology accessed using TEM from WT, MIC13 KO, SLP2 KO, MIC13-SLP2 DKO stably expressing pGIPZ-Control shRNA or pGIPZ-YME1L shRNA. Scale bar represents 0.5 μm. The skeletonization of the TEM image is depicted below the image. Mitochondria in MIC13 KO, SLP2 KO, MIC13-SLP2 DKO display loss of cristae and CJs, with cristae arranged as either stacks or concentric rings. SLP2 KO additionally display swollen cristae. YME1L depletion showed beneficial consequences on cristae morphology with presence of nascent CJs (red arrows). This shows that loss of CJs in MIC13 KO is attributed to MIC10 loss, and swelling of SLP2 KO cristae is visibly restored upon YME1L depletion. (E) Quantification of crista and CJs per mitochondrial section. Outliers were removed with Grubbs’ method and statistical significance was analyzed using Student’s t test. ∗ p -value ≤0.05, ∗∗ p -value ≤0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p -value ≤0.0001, ns = non-significant, P-value >0.05.

Article Snippet: Blocking solution was removed and primary antibodies with 1:100 dilution ratio was added to the samples and incubation was carried out at 37°C for 2 h. The following primary antibodies were used: MIC10 (Abcam, 84969), MIC13 (custom made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625-1-AP), MIC25 (Proteintech, 20639-1-AP), MIC26 (Thermofisher Scientific, MA5-15493), MIC27 (Sigma-Aldrich, HPA000612-100UL), MIC60 (Abcam, ab110329), SLP2 (Abcam, ab102051), SLP2 (OriGene, TA808240), Mt-CO2 (Abcam, ab110258)), OPA1 (custom-made, Pineda against human OPA1 using peptide CDLKKVREIQEKLDAFIEALHQEK, Vinculin (Sigma-Aldrich, V9131).

Techniques: Western Blot, Stable Transfection, Expressing, Control, shRNA, Isolation

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: Schematic illustration of synergistic role of SLP2 and MIC13 in MICOS assembly and crista junction formation The schematic model illustrates the quality control processes involved in cristae junction (CJ) morphogenesis and MICOS homeostasis, mediated by the MIC13-YME1L and SLP2-YME1L, which differentially stabilize components of the MIC10-subcomplex. This process facilitates the formation of the “seeder complex”, which consists of SLP2 and the stabilized MIC10-subcomplex. The simultaneous depletion of MIC13 and SLP2 disrupts the nanoscale distribution of MIC60 and its integration into the MICOS complex. The proposed “seeder model” suggests that the formation of the seeder complex is crucial for organizing the punctate distribution of MIC60 and facilitating its assembly into the MICOS-MIB complex. This process is essential for the formation of nascent CJs and establishing contact sites between IM and OM. In addition to identifying SLP2 as a regulator of cristae morphology, this model provides key insights into the intricate and partially overlapping quality control pathways governing MICOS regulation, elucidates specific functions attributed to MIC13, and highlights the interdependency between the MIC10- and MIC60-subcomplexes in MICOS-MIB complex assembly.

Article Snippet: Blocking solution was removed and primary antibodies with 1:100 dilution ratio was added to the samples and incubation was carried out at 37°C for 2 h. The following primary antibodies were used: MIC10 (Abcam, 84969), MIC13 (custom made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625-1-AP), MIC25 (Proteintech, 20639-1-AP), MIC26 (Thermofisher Scientific, MA5-15493), MIC27 (Sigma-Aldrich, HPA000612-100UL), MIC60 (Abcam, ab110329), SLP2 (Abcam, ab102051), SLP2 (OriGene, TA808240), Mt-CO2 (Abcam, ab110258)), OPA1 (custom-made, Pineda against human OPA1 using peptide CDLKKVREIQEKLDAFIEALHQEK, Vinculin (Sigma-Aldrich, V9131).

Techniques: Control

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet:

Article Snippet: Blocking solution was removed and primary antibodies with 1:100 dilution ratio was added to the samples and incubation was carried out at 37°C for 2 h. The following primary antibodies were used: MIC10 (Abcam, 84969), MIC13 (custom made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625-1-AP), MIC25 (Proteintech, 20639-1-AP), MIC26 (Thermofisher Scientific, MA5-15493), MIC27 (Sigma-Aldrich, HPA000612-100UL), MIC60 (Abcam, ab110329), SLP2 (Abcam, ab102051), SLP2 (OriGene, TA808240), Mt-CO2 (Abcam, ab110258)), OPA1 (custom-made, Pineda against human OPA1 using peptide CDLKKVREIQEKLDAFIEALHQEK, Vinculin (Sigma-Aldrich, V9131).

Techniques: Virus, Recombinant, Protease Inhibitor, In Situ, Clone Assay, Transfection, Mass Spectrometry, Knock-Out, Double Knockout, Stable Transfection, Expressing, Plasmid Preparation, Control, shRNA, CRISPR, Software