rabbit monoclonal anti scp4 cell signaling technology  (Cell Signaling Technology Inc)

Journal: Cell reports

Article Title: SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia.

doi: 10.1016/j.celrep.2021.110233

Figure Lengend Snippet: Figure 4. STK35 and PDIK1L bind selectively to the catalytically active form of SCP4 (A) Western blot of cytoplasm and nuclear fractions from MOLM-13 cells. (B) Representative FLAG-SCP4 affinity purification western blot analysis for the subsequent mass spectrometry (MS) analysis. Cytoplasm and nucleus fractions from MOLM-13 cells stably expressing empty vector, FLAG-SCP4 WT, or catalytic mutant (D293A). Nuclear fraction was mixed with anti-FLAG M2 agarose overnight. The flow-through was analyzed to ensure efficient binding of the FLAG-tagged constructs (loaded as ‘‘unbound’’). Following extensive washing, the agarose amount equivalent to the cytoplasm and nucleus fractions loading was boiled in Laemmli buffer and loaded as ‘‘FLAG-IP.’’ The rest was sent for the MS analysis. (C) Venn diagram depicting the overlap between proteins detected by MS and absent in empty vector controls in the 2 independent biological replicates. (D) Total unique peptide counts for SCP4, PDIK1L, and STK35 detected by MS in the 2 independent biological replicates. (E) Domain architectures and homology heat-map of human STK35 and PDIK1L. ATP-BS, ATP-binding site. (F–H) Immunoprecipitation followed by western blotting performed with the indicated antibodies. The whole-cell lysate was prepared from HEK293T 24 h post- transfection with the indicated constructs (F). The nuclear lysates were prepared from the human MOLM-13 cells stably expressing the indicated constructs (G and H). –, empty vector; WT, wild-type FLAG-SCP4; 236–466, FLAG-SCP4236466; D293A, catalytic mutant FLAG-SCP4D293A; IP, immunoprecipitation. Note: degradation bands appear in the WT and D293A input at 50 kDa and in D293A at 40 kDa. See also Figure S4.

Article Snippet: Antibodies used in this study included SCP4 (CTDSPL2) (CST, # 6932, 1:500), FLAG (Sigma Aldrich, F1804, 1:10,000), HA-HRP (Sigma Aldrich, clone 3F10, 1:10,000), H3K4me3 (Sigma Aldrich, 07-473, 1:1,000), b-Actin-HRP (Sigma A3854-200UL; 1:50,000).

Techniques: Western Blot, Mass Spectrometry, Stable Transfection, Expressing, Plasmid Preparation, Mutagenesis, Binding Assay, Construct, Immunoprecipitation, Transfection

Journal: Cell reports

Article Title: SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia

doi: 10.1016/j.celrep.2021.110233

Figure Lengend Snippet: (A) Competition-based proliferation assays in 23 human cancer cell lines infected with the indicated sgRNAs. PDAC, pancreatic ductal adenocarcinoma; RMS, rhabdomyosarcoma. n = 3. (B) Western blot of whole-cell lysates from MOLM-13 cells on day 5 post-infection with the indicated sgRNAs. (C) Competition-based proliferation assay in MOLM-13 cells infected with the indicated sgRNAs. n = 3. (D) Western blot of whole-cell lysates from K562 cells on day 5 post-infection with the indicated sgRNAs. (E) Competition-based proliferation assay in K562 cells infected with the indicated sgRNAs. n = 3. (F) Quantification of the different cell-cycle stages in MOLM-13 cells on day 5 post-infection with the indicated sgRNAs. n = 3. (G) Bioluminescence imaging of NSG mice transplanted with luciferase + /Cas9 + MOLM-13 cells infected with either sgROSA or sgSCP4. (H) Quantification of bioluminescence intensity. n = 3. p value was calculated by unpaired Student’s t-test. *p < 0.05. (I) Western blot of whole-cell lysates from CD34 + cells electroporated with Cas9 loaded with the indicated sgRNAs on day 8 post-electroporation, with culturing in myeloid conditions. (J) qRT-PCR analysis of indels presence in CD34 + cells electroporated with Cas9 loaded with the indicated sgRNAs over the course of culturing in myeloid conditions. The effects of individual sgRNAs for SCP4 were averaged. n = 4. (K) Quantification of the flow cytometry analysis of myeloid differentiation of CD34 + cells electroporated with Cas9 loaded with the indicated sgRNAs on day 8 post-electroporation, with culturing in myeloid conditions. The effects of individual negative controls and sgRNAs for SCP4 were averaged. n = 4. All bar graphs represent the mean ± SEM. All sgRNA experiments were performed in Cas9-expressing cell lines. “e” refers to the exon number targeted by each sgRNA. The indicated sgRNAs were linked to GFP. ROSA, Mock, and NT, negative controls; PCNA and MYC, positive controls. See also .

Article Snippet: Antibodies used in this study included SCP4 ( CTDSPL2 ) (CST, # 6932, 1:500), FLAG (Sigma Aldrich, F1804, 1:10,000), HA-HRP (Sigma Aldrich, clone 3F10, 1:10,000), H3K4me3 (Sigma Aldrich, 07–473, 1:1,000), β-Actin-HRP (Sigma A3854-200UL; 1:50,000).

Techniques: Infection, Western Blot, Proliferation Assay, Imaging, Luciferase, Electroporation, Quantitative RT-PCR, Flow Cytometry, Expressing

Journal: Cell reports

Article Title: SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia

doi: 10.1016/j.celrep.2021.110233

Figure Lengend Snippet: (A) Relative evolutionary conservation score for each residue from 0 — least conserved to 1 — most conserved. Based on data from . (B) Protein disorder prediction score for each residue from 0 — least disordered to 1 — most disordered. Based on data from . (C) Running average of log 2 fold changes of the CRISPR scan of SCP4 with all the possible sgRNAs. SCP4 protein amino acid numbers are indicated along the x axis. (D) Domain architectures of human SCP4 and the truncated version of SCP4 used in this study. (E) Western blot of whole-cell lysates from MOLM-13 cells stably expressing empty vector or CRISPR-resistant SCP4 236–466 . (F) Competition-based proliferation assay in MOLM-13 cells stably expressing empty vector or CRISPR-resistant SCP4 236–466 infected with the indicated sgRNAs. n = 3. (G) Western blot of whole-cell lysates from MOLM-13 cells stably expressing empty vector (EV; underloaded) or CRISPR-resistant wild type (WT) and catalytic mutants of SCP4. Vertical black dashed lines indicate omitted lanes from the same gel and same exposure. (H) Competition-based proliferation assay in MOLM-13 cells stably expressing empty vector or CRISPR-resistant WT and catalytic mutants of SCP4 infected with the indicated sgRNAs. n = 3. All bar graphs represent the mean ± SEM. ROSA, negative control; PCNA, positive control. See also and .

Article Snippet: Antibodies used in this study included SCP4 ( CTDSPL2 ) (CST, # 6932, 1:500), FLAG (Sigma Aldrich, F1804, 1:10,000), HA-HRP (Sigma Aldrich, clone 3F10, 1:10,000), H3K4me3 (Sigma Aldrich, 07–473, 1:1,000), β-Actin-HRP (Sigma A3854-200UL; 1:50,000).

Techniques: Residue, CRISPR, Western Blot, Stable Transfection, Expressing, Plasmid Preparation, Proliferation Assay, Infection, Negative Control, Positive Control

Journal: Cell reports

Article Title: SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia

doi: 10.1016/j.celrep.2021.110233

Figure Lengend Snippet: (A) Western blot of cytoplasm and nuclear fractions from MOLM-13 cells. (B) Representative FLAG-SCP4 affinity purification western blot analysis for the subsequent mass spectrometry (MS) analysis. Cytoplasm and nucleus fractions from MOLM-13 cells stably expressing empty vector, FLAG-SCP4 WT, or catalytic mutant (D293A). Nuclear fraction was mixed with anti-FLAG M2 agarose overnight. The flow-through was analyzed to ensure efficient binding of the FLAG-tagged constructs (loaded as “unbound”). Following extensive washing, the agarose amount equivalent to the cytoplasm and nucleus fractions loading was boiled in Laemmli buffer and loaded as “FLAG-IP.” The rest was sent for the MS analysis. (C) Venn diagram depicting the overlap between proteins detected by MS and absent in empty vector controls in the 2 independent biological replicates. (D) Total unique peptide counts for SCP4, PDIK1L, and STK35 detected by MS in the 2 independent biological replicates. (E) Domain architectures and homology heat-map of human STK35 and PDIK1L. ATP-BS, ATP-binding site. (F–H) Immunoprecipitation followed by western blotting performed with the indicated antibodies. The whole-cell lysate was prepared from HEK293T 24 h post-transfection with the indicated constructs (F). The nuclear lysates were prepared from the human MOLM-13 cells stably expressing the indicated constructs (G and H). –, empty vector; WT, wild-type FLAG-SCP4; 236–466, FLAG-SCP4 236–466 ; D293A, catalytic mutant FLAG-SCP4 D293A ; IP, immunoprecipitation. Note: degradation bands appear in the WT and D293A input at ~50 kDa and in D293A at ~40 kDa. See also .

Article Snippet: Antibodies used in this study included SCP4 ( CTDSPL2 ) (CST, # 6932, 1:500), FLAG (Sigma Aldrich, F1804, 1:10,000), HA-HRP (Sigma Aldrich, clone 3F10, 1:10,000), H3K4me3 (Sigma Aldrich, 07–473, 1:1,000), β-Actin-HRP (Sigma A3854-200UL; 1:50,000).

Techniques: Western Blot, Affinity Purification, Mass Spectrometry, Stable Transfection, Expressing, Plasmid Preparation, Mutagenesis, Binding Assay, Construct, Immunoprecipitation, Transfection

Journal: Cell reports

Article Title: SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia

doi: 10.1016/j.celrep.2021.110233

Figure Lengend Snippet: (A and B) Western blot of cytoplasm (Cyto) and nucleus (Nucl) fractions of MOLM-13 cells stably expressing HA-PDIK1L (A) or HA-STK35 (B) on day 5 post-infection with the indicated sgRNAs. Shown is a representative experiment of 3 independent biological replicates. (C) Schematics of phosphorylation sites reported in the publicly available phospho-proteomics datasets on STK35 and PDK1L relative to their domain architectures ( ; ). aa #, amino acid number; P, phosphorylation; ATP-BS, ATP-binding site. (D) Phosphorylated peptides assayed for SCP4 phosphatase activity in vitro . (E) Recombinant SCP4 protein purity as assessed by SDS-PAGE and Coomassie blue staining. His-SUMO-(TEV)-SCP4 was expressed in BL21 (DE3) cells and purified by affinity, anion exchange, and gel filtration chromatography. Molecular weight markers are shown for reference. (F and G) Phosphatase activity of SCP4 against the indicated peptides plotted for kinetic fitting. Each measurement was conducted in triplicate with standard deviations shown as error bars. (H) Competition-based proliferation assay in MOLM-13 cells stably expressing EV or the CR HA-PDIK1L or HA-STK35 constructs harboring the indicated amino acid substitutions infected with the indicated sgRNAs. n = 3. (I) Western blot of whole-cell lysates from MOLM-13 cells stably expressing EV or CR HA-PDIK1L or HA-STK35 constructs harboring the indicated amino acid substitutions. All bar graphs represent the mean ± SEM. See also .

Article Snippet: Antibodies used in this study included SCP4 ( CTDSPL2 ) (CST, # 6932, 1:500), FLAG (Sigma Aldrich, F1804, 1:10,000), HA-HRP (Sigma Aldrich, clone 3F10, 1:10,000), H3K4me3 (Sigma Aldrich, 07–473, 1:1,000), β-Actin-HRP (Sigma A3854-200UL; 1:50,000).

Techniques: Western Blot, Stable Transfection, Expressing, Infection, Phospho-proteomics, Binding Assay, Activity Assay, In Vitro, Recombinant, SDS Page, Staining, Purification, Filtration, Chromatography, Molecular Weight, Proliferation Assay, Construct

Journal: Cell reports

Article Title: SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia

doi: 10.1016/j.celrep.2021.110233

Figure Lengend Snippet: (A) Venn diagram depicting the overlap between statistically significant downregulated genes in MOLM-13 cells upon SCP4 knockout and STK35/PDIK1L double knockout. DeSeq2 (n = 4). (B) Ontology analysis of overlapping statistically significant downregulated genes in MOLM-13 cells upon both SCP4 knockout and STK35/PDIK1L double knockout. (C) Selected statistically significant downregulated genes in MOLM-13 cells upon both SCP4 knockout and STK35/PDIK1L double knockout and the amino acids they are involved in biosynthesis or transport of. (D) The correlation between log2 fold changes in the levels of selected metabolites upon SCP4 knockout and STK35/PDIK1L double knockout relative to negative control as measured by the MS analysis. Every dot represents the mean ± SEM (n = 6). Amino acids are in red, and there are a few unchanged metabolites in black for reference. Shown is a representative experiment of 3 independent biological replicates. (E) Model. See also .

Article Snippet: Antibodies used in this study included SCP4 ( CTDSPL2 ) (CST, # 6932, 1:500), FLAG (Sigma Aldrich, F1804, 1:10,000), HA-HRP (Sigma Aldrich, clone 3F10, 1:10,000), H3K4me3 (Sigma Aldrich, 07–473, 1:1,000), β-Actin-HRP (Sigma A3854-200UL; 1:50,000).

Techniques: Knock-Out, Double Knockout, Negative Control

Journal: Cell reports

Article Title: SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia

doi: 10.1016/j.celrep.2021.110233

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibodies used in this study included SCP4 ( CTDSPL2 ) (CST, # 6932, 1:500), FLAG (Sigma Aldrich, F1804, 1:10,000), HA-HRP (Sigma Aldrich, clone 3F10, 1:10,000), H3K4me3 (Sigma Aldrich, 07–473, 1:1,000), β-Actin-HRP (Sigma A3854-200UL; 1:50,000).

Techniques: Virus, Recombinant, Lysis, Plasmid Preparation, Magnetic Beads, Cloning, Purification, Bicinchoninic Acid Protein Assay, SYBR Green Assay, Gel Extraction, Ligation, Sample Prep, Mass Spectrometry, Software

Journal: Cell reports

Article Title: SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia

doi: 10.1016/j.celrep.2021.110233

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-SCP4 , Cell Signaling Technology , Cat# 6932.

Techniques: Virus, Recombinant, Lysis, Plasmid Preparation, Magnetic Beads, Cloning, Purification, Bicinchoninic Acid Protein Assay, SYBR Green Assay, Gel Extraction, Ligation, Sample Prep, Mass Spectrometry, Software