Journal: Molecular Therapy Oncology

Article Title: SAP30, a novel autophagy regulatory gene in neuroblastoma

doi: 10.1016/j.omton.2024.200916

Figure Lengend Snippet: Silencing of SAP30 inhibits autophagy in NB cells (A and B) Western blot analysis illustrates the protein levels of SAP30, p62, LC3B-I, and LC3B-II in SAP30-silenced stable SK-N-AS, NB-19, and SK-N-BE(2) cells (A) or transfection with SAP30-specific siRNAs in SK-N-AS, and NB-19 cells (B) for 48 h. (C) Immunofluorescence images show the expression levels of LC3B and p62 in SAP30-silenced stable SK-N-AS cells for 48 h. LC3B was visualized using an anti-LC3B-AF488 (green) antibody, while p62 was detected using an anti-p62-AF647 (red) antibody. (D) Immunofluorescence images depict the expression levels of GFP-LC3B in stable SK-N-AS cells with silenced SAP30. The cells were transfected with GFP-LC3B or GFP-empty vector (EV) for 48 h, followed by immunofluorescence analysis using a GFP antibody.

Article Snippet: Primary antibodies employed in this study included SAP30 (Novus Biologicals, catalog no. NBP1-92648), p62/SQSTM1 (Proteintech, catalog no. 66184-1-Ig), LC3B (Proteintech, catalog no. 14600-1-AP), Syntaxin 17 (Proteintech, catalog no. 17815-1-AP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Proteintech, catalog no. 60004-1-Ig).

Techniques: Western Blot, Transfection, Immunofluorescence, Expressing, Plasmid Preparation

Journal: Molecular Therapy Oncology

Article Title: SAP30, a novel autophagy regulatory gene in neuroblastoma

doi: 10.1016/j.omton.2024.200916

Figure Lengend Snippet: Silencing of SAP30 disrupts starvation-induced physiological autophagy in NB cells (A and B) Western blot analysis of SAP30, p62, LC3B-I, and LC3B-II protein levels in SAP30-silenced stable NB-19 (A) and SK-N-AS (B) cell lines following 12 and 24 h of serum starvation. SFM, serum-free medium; SRM, serum-rich medium. (C) Flow cytometry plots illustrate the degradation of GFP-LC3B in SAP30-silenced stable SK-N-AS cell lines after 4 h of amino acid and serum starvation using HBSS. Stable NB cell lines were transiently transfected with the GFP-LC3B-RFP-LC3BΔG plasmid before starvation, and the normalized GFP/RFP ratio, an indicator of LC3B degradation, is provided. (D and E) Western blot analysis of p62 and LC3B-II protein levels in SAP30-silenced stable SK-N-B(E)2 (D), SK-N-AS, and NB-19 (E) cell lines after treatment with the lysosome-targeting autophagy inhibitor bafilomycin (5 nM) for 24 h. (F) Immunofluorescence images depict the co-localization of LC3B and LAMP-1 in SAP30-silenced stable SK-N-AS cells following 4 h of HBSS starvation. LC3B was detected with an anti-LC3B-Alexa Fluor 488 antibody (green), while LAMP-1 was visualized using an anti-LAMP-1-Alexa Fluor 568 antibody (red). DAPI staining in blue was used to mark the nucleus. White arrows indicate inhibited colocalization.

Article Snippet: Primary antibodies employed in this study included SAP30 (Novus Biologicals, catalog no. NBP1-92648), p62/SQSTM1 (Proteintech, catalog no. 66184-1-Ig), LC3B (Proteintech, catalog no. 14600-1-AP), Syntaxin 17 (Proteintech, catalog no. 17815-1-AP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Proteintech, catalog no. 60004-1-Ig).

Techniques: Western Blot, Flow Cytometry, Transfection, Plasmid Preparation, Immunofluorescence, Staining

Journal: Molecular Therapy Oncology

Article Title: SAP30, a novel autophagy regulatory gene in neuroblastoma

doi: 10.1016/j.omton.2024.200916

Figure Lengend Snippet: SAP30 ectopic expression induces physiological autophagy in NB cells under starvation conditions (A and B) Western blot analysis of p62, LC3B-I, and LC3B-II protein levels in SAP30-overexpressing stable SK-N-AS (A and C) and NB-19 (B and D) cell lines after 2 and 4 h of amino acid starvation using HBSS (A and B) or cultured in medium containing different percentages of FBS (C and D) and treatment with or without the autophagy inhibitor bafilomycin (5 nM) for 24 h. The fold change in the expression levels of p62 and LC3B-II proteins normalized to GAPDH is provided below the respective western blot images. (E–G) Immunofluorescence images depict the expression of LC3B in SAP30-overexpressing stable NB-19 (E and F) and SK-N-AS (G) cell lines under different starvation conditions. Cells were starved with HBSS (E and G) or SFM (F) and treated with bafilomycin or hydroxychloroquine (HCQ). LC3B was detected using an anti-LC3B-Alexa Fluor 568 antibody (red), and DAPI staining (blue) was used to label the nucleus. The fold change in expression levels of p62 and LC3B-II proteins, normalized to GAPDH, is provided below the respective western blot images. HE, higher exposure; LE, lower exposure.

Article Snippet: Primary antibodies employed in this study included SAP30 (Novus Biologicals, catalog no. NBP1-92648), p62/SQSTM1 (Proteintech, catalog no. 66184-1-Ig), LC3B (Proteintech, catalog no. 14600-1-AP), Syntaxin 17 (Proteintech, catalog no. 17815-1-AP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Proteintech, catalog no. 60004-1-Ig).

Techniques: Expressing, Western Blot, Cell Culture, Immunofluorescence, Staining

Journal: Molecular Therapy Oncology

Article Title: SAP30, a novel autophagy regulatory gene in neuroblastoma

doi: 10.1016/j.omton.2024.200916

Figure Lengend Snippet: The stability of SAP30 and its degradation mechanisms independent of autophagy during starvation (A and B) Western blot analysis of p62, SAP30, LC3B-I, and LC3B-II protein levels in SK-N-AS (A) and NB-19 (B) cells following 4 h of amino acid starvation using HBSS, and treatment with bafilomycin (50 nM), the proteasomal inhibitor MG132 (1 μM), and HCQ (50 μM). (C) Immunofluorescence analysis depicts the localization of p62 (red) in the cytosol and SAP30 (green) in both the nucleus and cytosol in SK-N-AS cells after 4 h of HBSS starvation, with or without treatment with bafilomycin (50 nM). (D and E) Western blot analysis of p62, SAP30, LC3B-I, and LC3B-II protein levels in SK-N-AS (D) and NB-19 (E) cells after 24 or 9 h of starvation, respectively, using SFM (0% FBS) in the presence of bafilomycin or HCQ. (F and G) Western blot analysis of p62, SAP30, LC3B-I, and LC3B-II protein levels in SK-N-AS (F) and NB-19 (G) cells after 48 h of starvation using reduced serum (1% FBS) medium in the presence of bafilomycin (2.5 nM), HCQ (25 μM), or MG132 (0.5 μM). The fold change in expression levels of p62 and SAP30 proteins, normalized to GAPDH, is provided below the respective western blot images.

Article Snippet: Primary antibodies employed in this study included SAP30 (Novus Biologicals, catalog no. NBP1-92648), p62/SQSTM1 (Proteintech, catalog no. 66184-1-Ig), LC3B (Proteintech, catalog no. 14600-1-AP), Syntaxin 17 (Proteintech, catalog no. 17815-1-AP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Proteintech, catalog no. 60004-1-Ig).

Techniques: Western Blot, Immunofluorescence, Expressing

Journal: Molecular Therapy Oncology

Article Title: SAP30, a novel autophagy regulatory gene in neuroblastoma

doi: 10.1016/j.omton.2024.200916

Figure Lengend Snippet: Silencing of SAP30 reduces STX17 expression and its availability for translocation to the autophagic membrane (A) Real-time qPCR (left) and western blot (center) analyses display the mRNA and protein levels of SAP30, STX17, and ATG14 in stable NB-19 and SK-N-AS cells with silenced SAP30. A western blotting densitometric quantification graph shows the fold change in expression levels of STX17, normalized to GAPDH protein (right). (B and C) Immunofluorescence images depict the expression levels of STX17 in stable NB-19 (B) and SK-N-AS (C) cells with silenced SAP30 following 4 h of starvation using HBSS in the presence of bafilomycin (50 nM). Polyclonal rabbit antibody against STX17 was utilized, followed by immunofluorescence analysis using secondary antibody labeled with Alexa Fluor 488 (green). (D and E) Confocal microscopy images display the co-localization of STX17 and LC3B in stable SK-N-AS cells with silenced SAP30 following 4 h of HBSS incubation along with bafilomycin (50 nM) (D) and HCQ (50 μM) (E). Antibodies against STX17 and LC3B-II were employed, followed by immunofluorescence analysis using secondary antibodies labeled with Alexa Fluor 488 (green) and AF568 (red), respectively. The magnified confocal microscopy images illustrate the orange-yellow colocalized dots of STX17 (green) and LC3B-II (red) resulting from the combination of the two channels in cells. (F) Western blot analysis of p62, LC3B-I, and LC3B-II protein levels in stable SK-N-AS cells with silenced SAP30 following 48 h of transfection with FLAG-EV or FLAG-STX17 overexpression plasmids and with or without 4 h of serum and amino acid starvation using HBSS. Bafilomycin and HCQ were added with HBSS. Densitometric quantification numbers show the fold change in expression levels of p62 and LC3B-II, normalized to GAPDH, at the bottom of the western blot images.

Article Snippet: Primary antibodies employed in this study included SAP30 (Novus Biologicals, catalog no. NBP1-92648), p62/SQSTM1 (Proteintech, catalog no. 66184-1-Ig), LC3B (Proteintech, catalog no. 14600-1-AP), Syntaxin 17 (Proteintech, catalog no. 17815-1-AP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Proteintech, catalog no. 60004-1-Ig).

Techniques: Expressing, Translocation Assay, Membrane, Western Blot, Immunofluorescence, Labeling, Confocal Microscopy, Incubation, Transfection, Over Expression

Journal: Molecular Therapy Oncology

Article Title: SAP30, a novel autophagy regulatory gene in neuroblastoma

doi: 10.1016/j.omton.2024.200916

Figure Lengend Snippet: SAP30 promotes cell growth and autophagy protection under therapeutic stress in NB cells (A and B) Quantification graphs depict live cell counts analyzed using the trypan blue exclusion method in stable SK-N-AS (A) and NB-19 (B) cells with silenced SAP30, cultured in either 10% FBS medium (FM) or 1% FBS medium (1% FBS M) for 4 days. (C and D) Flow cytometry plots illustrate the distribution of NB cells (%) in the G1-, S-, and G2-phases upon SAP30 silencing. Stable SK-N-AS (C) and NB-19 (D) cells with silenced SAP30 were cultured in either 10% FM or 1% FBS M for 2 days, followed by apoBrdU-allophycocyanin (APC) and 7-amino-actinomycin D (7-AAD) staining. Quantification graphs display the cells (%) in the S-phase on the right side of the flow cytometry graphs. (E and F) Flow cytometry plots depict the intracellular expression of LC3B in SK-N-AS (E) and NB-19 (F) cells with stable expression of either FLAG-EV or FLAG-SAP30, treated with doxorubicin (0.5 μg/mL) alone or in combination with bafilomycin (2.5 nM) and HCQ (25 μM) for 48 h. (G) Cell viability quantification graphs, analyzed by MTT assay in SK-N-AS, NB-19 cells with stable expression of either FLAG-EV or FLAG-SAP30, treated with doxorubicin (0.5 μg/mL) or cisplatin (2.5 μg/mL) alone or in combination with HCQ (25 μM) for 48 h. These experiments were independently replicated three times, with error bars representing the SE. Statistical comparisons were conducted using a two-tailed unpaired Student’s t test, and significance levels were indicated as ∗∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05, respectively.

Article Snippet: Primary antibodies employed in this study included SAP30 (Novus Biologicals, catalog no. NBP1-92648), p62/SQSTM1 (Proteintech, catalog no. 66184-1-Ig), LC3B (Proteintech, catalog no. 14600-1-AP), Syntaxin 17 (Proteintech, catalog no. 17815-1-AP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Proteintech, catalog no. 60004-1-Ig).

Techniques: Cell Culture, Flow Cytometry, Staining, Expressing, MTT Assay, Two Tailed Test

Journal: Molecular Therapy Oncology

Article Title: SAP30, a novel autophagy regulatory gene in neuroblastoma

doi: 10.1016/j.omton.2024.200916

Figure Lengend Snippet: Silencing SAP30 results in increased accumulation of LC3B and reduced levels of SAP30-responsive STX17 in NB, both in vivo and in PDX tumors The IHC images depict the expression levels of (A) LC3B (top), STX17 (center), and Ki67 (bottom), in tumors from NSG mice injected with stable SK-N-B(E)2 cells expressing either control shRNA or two different SAP30-specific shRNAs. (B) Additionally, LC3B expression in PDX tumors from NB patients at diagnosis and relapse stages is shown. The intensity of LC3B staining in the cytoplasm of each cell/frame was quantified using HALO software to compute an H-score. Protein expression is indicated by brown staining, while blue staining represents the nucleus. Images were captured at 20× magnification, with enlarged images provided at 40× magnification in the lower right corner. Scale bar: 75 μm. A two-way ANOVA denotes statistical significance levels as ∗∗∗∗ p < 0.001.

Article Snippet: Primary antibodies employed in this study included SAP30 (Novus Biologicals, catalog no. NBP1-92648), p62/SQSTM1 (Proteintech, catalog no. 66184-1-Ig), LC3B (Proteintech, catalog no. 14600-1-AP), Syntaxin 17 (Proteintech, catalog no. 17815-1-AP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Proteintech, catalog no. 60004-1-Ig).

Techniques: In Vivo, Expressing, Injection, Control, shRNA, Biomarker Discovery, Staining, Software

Journal: PLoS Pathogens

Article Title: A SAP30 Complex Inhibits IFN-β Expression in Rift Valley Fever Virus Infected Cells

doi: 10.1371/journal.ppat.0040013

Figure Lengend Snippet: (A) L929 wt330 cells, carrying an integrated wild type muIFN-β promoter fused to CAT reporter gene, were mock infected or infected by RVFV ZH or C13 or with NDV. Total cell extracts were prepared at 4, 6 and 8 h p.i. and CAT actvity was measured. (B) Non-confocal conventional fluorescence microscopy was used to analyze the nuclear distribution of NSs filaments in murine L929 cells infected by C13 or ZH. Presence of NSs filament detected using rabbit polyclonal anti-NSs antibody (green) or total DNA distribution revealed with Hoechst 33258 are shown respectively, in left and middle panels. Merged images are shown in right panels. Scale bars, 10 μm. (C) For yeast two-hybrid screening, AH109 yeast were co-transformed by pACT2-SAP30 1–152 that expressed Gal4 transactivating domain fused to the open reading frames corresponding to the 152 first aa of SAP30 and pGBKT7, pGBKT7-NSs ZH , pGBKT7-NSs C13 , pGBKT7-NSs TOS , or pGBKT7-NSs GER in which NSs from RVFV ZH or C13 or NSs proteins from Toscana (TOSV) and Germiston (GERV) bunyaviruses were fused to the Gal4 DNA-binding domain. The values of β galactosidase activity represent at least four independent experiments with SD bars. (D) Two-hybrid system using full length wild type NSs ZH or the deleted forms. The numbers indicate the amino acid position in the reference sequence. The sequence lacking amino acids 16–198 correspond to C13. (E) GST-NSs (left panel) or GST-SAP30 (right panel) was incubated with an extract from 293 cells transfected with the HA tagged-SAP30 expressing plasmid (left panel) or from ZH infected L929 cells (right panel). After extensive washing, the proteins bound to the beads were analysed by western blots using antibodies against HA (left panel) or NSs (right panel). The Coomassie blue staining showing that equivalent amounts of GST fusion proteins were loaded on the beads is not shown. (F) HEK 293 cells were transfected with either pCS2-Myc (lanes 1,2) or pCS2-Myc-SAP30 (lane 3) and either not infected (lane 1) or infected with ZH (lanes 2,3). Cell lysates were precipitated with anti-myc (9E10) antibody. Crude lysates (input) and the precipitated proteins (IP) were detected with anti-myc and anti-NSs antibodies.

Article Snippet: The plasmid pCi-HA-SAP30 was constructed by inserting the HA-tagged full length murine SAP30 cassette from the pACT2 plasmid into the pCi vector at the BglII site (Promega).

Techniques: Infection, Fluorescence, Microscopy, Two Hybrid Screening, Transformation Assay, Binding Assay, Activity Assay, Sequencing, Incubation, Transfection, Expressing, Plasmid Preparation, Western Blot, Staining

Journal: PLoS Pathogens

Article Title: A SAP30 Complex Inhibits IFN-β Expression in Rift Valley Fever Virus Infected Cells

doi: 10.1371/journal.ppat.0040013

Figure Lengend Snippet: Colocalization of endogenous SAP30 (A, C, and E) and YY1 proteins (B, D, and F) with NSs filament was analyzed by confocal microscopy in L929 wt330 cells uninfected (NI) or infected with C13 or ZH at m.o.i. 5 collected at 18 h p.i. (A and B), 5h (C and D) or 7 h p.i. (E and F). Each row represents a single optical section of the same nucleus. A, C, and E) Left panels (a, d, g) correspond to SAP30 distribution revealed with goat polyclonal anti-SAP30 antibodies. Middle panels (b, e, h) show subnuclear NSs distribution detected with anti-NSs rabbit polyclonal antibodies. Merged images of SAP30 and NSs are shown on right panels (c, f, i). (B, D, and F) Left panels (a, d, g) correspond to YY1 distribution revealed with mouse monoclonal anti-YY1 antibody. Middle panels (b, e, h) show subnuclear NSs distribution detected with anti-NSs rabbit polyclonal antibody. Merged images of YY1 and NSs are shown on right panels (c, f, i). Scale bar, 10 μm.

Article Snippet: The plasmid pCi-HA-SAP30 was constructed by inserting the HA-tagged full length murine SAP30 cassette from the pACT2 plasmid into the pCi vector at the BglII site (Promega).

Techniques: Confocal Microscopy, Infection

Journal: PLoS Pathogens

Article Title: A SAP30 Complex Inhibits IFN-β Expression in Rift Valley Fever Virus Infected Cells

doi: 10.1371/journal.ppat.0040013

Figure Lengend Snippet: L929 cells (A) or L929 wt330 (B, C, and E), or L929 wt330, mut90, and mut122 (D) were infected with ZH or C13 at a m.o.i. of 5 and collected at 6 h p.i. or as indicated (C). Input and DNA immunoprecipitated (IP) with specific antibodies as indicated, were amplified with primers specific for the endogenous IFN-β promoter (A), for the integrated murine wild type wt330 IFN-β promoter (B-E), for the mutated integrated promoters (D) or for the murine β-actin gene (C). Schematic representation of murine IFN-β promoters either wild type (wt330) or mutated at the YY1 binding site present at position −90 (mut90) or −122 (mut122) is shown in D. Inputs are shown as controls except in E where they are the same as in D (wt330). Triangles indicate increasing amounts of DNA used during PCR reactions and corresponding in (B) to 1 μl, 2 μl, or 3 μl of 1:5 dilution of DNA immunoprecipitated with a-YY1 and a-SAP30 and of 1:50 dilution of DNA immunoprecipitated with a-NSs; (C) 1 μl and 2 μl of 1:50 dilution of DNA immunoprecipitated with a-NSs and 1 μl of 1:1,000 and 1:200 dilution of input DNA; (D) 1 μl of 1:5 and 1:1 dilution of DNA immunoprecipitated with a-YY1 and a-SAP30, 1 μl and 2 μl of 1:50 dilution of DNA immunoprecipitated with a-NSs and 1 μl of 1:1,000 and 1:200 dilution of input DNA.

Article Snippet: The plasmid pCi-HA-SAP30 was constructed by inserting the HA-tagged full length murine SAP30 cassette from the pACT2 plasmid into the pCi vector at the BglII site (Promega).

Techniques: Infection, Immunoprecipitation, Amplification, Binding Assay

Journal: PLoS Pathogens

Article Title: A SAP30 Complex Inhibits IFN-β Expression in Rift Valley Fever Virus Infected Cells

doi: 10.1371/journal.ppat.0040013

Figure Lengend Snippet: (A) Monolayers of Vero cells infected with RVFV rec-ZH or rec-ZHΔ210–230 were fixed and stained with crystal violet at 5 days p.i. (B) GST or GST-SAP30 was incubated with extracts from cells uninfected or infected with rec-ZH or rec-ZHΔ210–230. Proteins from the crude extracts (input) or after binding on GST or GST-NSs beads were analyzed by Western blotting using anti NSs antibodies. * denotes a cellular protein bound on GST-NSs which copurified with NSs. BF (C) or L929 wt 330 cells (D, E, F) uninfected or infected with C13, wt ZH, rec-ZH, or Δ210–230 were incubated for 8 h (C), 18 h (E) or for the indicated time (D and F). Extracts were prepared and analyzed by RT-PCR to detect C) IFN-β, GAPDH, or NSs mRNA as described in . (D) CAT activity, (E) confocal microscopy, or (F) chip experiment using anti-NSs antibodies like in ; (G) percentage of animals surviving after i.p. inoculation of 10 4 pfu.

Article Snippet: The plasmid pCi-HA-SAP30 was constructed by inserting the HA-tagged full length murine SAP30 cassette from the pACT2 plasmid into the pCi vector at the BglII site (Promega).

Techniques: Infection, Staining, Incubation, Binding Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Confocal Microscopy

Journal: PLoS Pathogens

Article Title: A SAP30 Complex Inhibits IFN-β Expression in Rift Valley Fever Virus Infected Cells

doi: 10.1371/journal.ppat.0040013

Figure Lengend Snippet: In uninfected cells, a SAP30/Sin3A/NCoR/HDAC-3 corepressor complex, described here for the first time, to our knowledge, interacts with the constitutively repressed murine IFN-β promoter through its YY1 binding site present at position −90. After RVFV ZH infection, in the presence of NSs filaments, recruitment of corepressor complex SAP30/Sin3A/NCoR/HDAC-3 is reinforced whereas recruitment of co-activator CBP and of YY1 at its −122 site as well as acetylation of histone residues K8H4 and K14H3 is inhibited. Therefore, IFN-β promoter remains silent in spite of IRF3 nuclear translocation and binding to the promoter.

Article Snippet: The plasmid pCi-HA-SAP30 was constructed by inserting the HA-tagged full length murine SAP30 cassette from the pACT2 plasmid into the pCi vector at the BglII site (Promega).

Techniques: Binding Assay, Infection, Translocation Assay

Journal: Cell reports

Article Title: SAP30BP interacts with RBM17/SPF45 to promote splicing in a subset of human short introns.

doi: 10.1016/j.celrep.2023.113534

Figure Lengend Snippet: Figure 1. SAP30BP is additionally required for RBM17-dependent splicing of pre-mRNAs with short introns (A) The model of RBM17-dependent splicing on a short intron with a truncated PPT (indicated by UUUUU).6 The associated splicing factors with the domain structures and the target sequences of pre-mRNAs are represented schematically. ‘‘Unknown factor X’’ was postulated in this study. (B) The depletion of RBM17, SAP30BP, and SAP30 proteins by siRNA-knockdown in HeLa cells. The depleted proteins were checked by western blotting with indicated antibodies. (C) In cellulo splicing assays of the pre-mRNAs including indicated introns. After the indicated siRNA transfection in HeLa cells, endogenous splicing of the four RBM17-dependent short introns and one RBM17-independent control intron were analyzed by RT-PCR. Means ± SEM are given for three independent experiments, and Welch’s t test values were calculated (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s. p > 0.05).

Article Snippet: Immuno-reactive protein bands were detected by the ECL system and visualized by imaging analyzer (ImageQuant LAS 500, GE Healthcare Life Sciences). siRNA knockdown and splicing assays siRNA-mediated knockdown in HeLa cells was performed as previously described.6 The siRNAs targeting RBM17, SAP30BP and SAP30 were purchased from Fasmac (Table S2 for the sequences).

Techniques: Knockdown, Western Blot, Transfection, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Cell reports

Article Title: SAP30BP interacts with RBM17/SPF45 to promote splicing in a subset of human short introns.

doi: 10.1016/j.celrep.2023.113534

Figure Lengend Snippet: Figure 3. UHM in RBM17 and newly found ULM in SAP30BP are critical for the binding between RBM17 and SAP30BP (A) Co-immunoprecipitation of RBM17 with SAP30BP and SF3B1. HeLa cells were co-transfected with Myc-SAP30BP, Myc-SAP30 (negative control), and FLAG-RBM17 plasmids; these transfected cell extracts were immunoprecipitated using mouse IgG or anti-FLAG antibody, and the immunoprecipitated proteins were analyzed by western blotting. The red arrowhead indicates IgG light chain of anti-FLAG antibody. (B) In vitro GST pull-down assay (after RNase A treatment) to detect the direct binding between indicated RBM17 variant proteins and SAP30BP protein. Red- colored variants indicate the diminished protein binding with SAP30BP. Recombinant SAP30BP protein associated with these GST-fused proteins was detected by western blotting using anti-SAP30BP antibody. The same membrane was also stained with Ponceau S to evaluate the loading amount and the comparable precipitation of the GST or GST fusion proteins.

Article Snippet: Immuno-reactive protein bands were detected by the ECL system and visualized by imaging analyzer (ImageQuant LAS 500, GE Healthcare Life Sciences). siRNA knockdown and splicing assays siRNA-mediated knockdown in HeLa cells was performed as previously described.6 The siRNAs targeting RBM17, SAP30BP and SAP30 were purchased from Fasmac (Table S2 for the sequences).

Techniques: Binding Assay, Immunoprecipitation, Transfection, Negative Control, Western Blot, In Vitro, Pull Down Assay, Variant Assay, Protein Binding, Recombinant, Membrane, Staining