human tsc2 flag tsc2 cdna  (Addgene inc)

Journal: iScience

Article Title: Growth factor-independent mTORC1 signaling promotes primary cilia length via suppression of autophagy

doi: 10.1016/j.isci.2025.114204

Figure Lengend Snippet: Genetic perturbation of mTORC1 signaling demonstrates that it promotes the elongation of primary cilia (A and B) Immunoblot (A) and primary cilia length (B) in doxycycline-inducible shScrambled and shMTOR RPE1 cells treated with doxycycline (0.5 μg/mL) for 7 days. (C) Immunoblot of isogenic clonal RPE1 cell lines with sgRNAs targeting TSC1, TSC2, or TBC1D7. Cells were serum-starved overnight. TSC1 and TSC2 are indicated by black arrowheads. (D) Primary cilia length in RPE1 lines used in (C). (E) Immunoblot of parental, sgTSC2, and human TSC2-rescued sgTSC2 RPE1 cells treated with Torin1 for 1 h. (F) Primary cilia length in RPE1 cell lines used in (E) following Torin1 treatment. (G) Immunoblot of Tsc2 +/+ , Tsc2 −/− , and human TSC2-rescued Tsc2 −/− MEFs after overnight serum starvation. (H) Primary cilia length in MEF cell lines used in (G). (I) Immunoblot of parental and sgNPRL2 RPE1 cells treated with overnight amino acid starvation followed by 1 h of amino acid stimulation. (J) Primary cilia length in parental and sgNPRL2 RPE1 cells treated with DMSO, Torin1, leucine deprivation, or complete amino acid deprivation. All treatments for primary cilia assessment were applied during the final 24 h in serum-free conditions (48 h total): DMSO (0.1%), rapamycin (20 nM), and Torin1 (250 nM). Data are represented as mean ± standard deviation (SD). Statistical analysis was performed using two-way ANOVA followed by Šídák’s multiple comparisons test. The significance threshold (α) was set at 0.05, and all p values are reported.

Article Snippet: Flag-tagged human TSC2 (Flag-TSC2) cDNA from pcDNA3 Flag TSC2 (Addgene; 14129) was PCR-amplified using Phusion High-Fidelity DNA Polymerase (Thermo Scientific; F531S) with primers containing sequences homologous to the insertion site in pMXS-IRES-Blast, a retroviral vector (Cell Biolabs, Inc; RTV-106): 5’-CTAGCTAGTTAATTAAatggactacaaagacgatgacgataaagc-3’ 5’-CGCCGGCCCTCGAGtcacacaaactcggtgaagtcctcc-3’ (Uppercase: homologous to pMXS-IRES-Blast; lowercase: homologous to Flag-TSC2).

Techniques: Western Blot, Standard Deviation

Journal: iScience

Article Title: Growth factor-independent mTORC1 signaling promotes primary cilia length via suppression of autophagy

doi: 10.1016/j.isci.2025.114204

Figure Lengend Snippet: mTORC1 signaling promotes elongation of primary cilia via autophagy suppression (A) Schematic diagram showing the generation of the GFP-LC3B-RFP autophagy reporter RPE1 cell line and flow cytometry analysis used to assess autophagic flux. (B and C) Flow cytometric analysis (B) and quantification of autophagic flux (C) in RPE1 cells treated with FBS (10%), bafilomycin A1, rapamycin, Torin1, or leucine deprivation for the indicated times. (D and E) Immunoblot (D) and primary cilia length (E) in RPE1 cells treated with DMSO, rapamycin, chloroquine, or bafilomycin A1. (F and G) Immunoblot (F) and primary cilia length (G) in RPE1 cells treated with DMSO, chloroquine, or bafilomycin A1, alone or in combination with rapamycin. (H) Primary cilia length in RPE1 cells treated with DMSO, ULK1/2 inhibitor (SBP-7455), or vacuoar protein sorting 34, class III PI 3-kinase (VPS34) inhibitor (VPS34-IN1). (I) Immunoblot of parental and sgATG7 RPE1 cells treated with bafilomycin A1 for the final 3 h under overnight serum starvation. p62 is indicated by a black arrowhead. (J) Primary cilia length in RPE1 cells used in (I) following treatment with DMSO, Torin1, or bafilomycin A1. (K) Immunoblot of parental and sgTFEB RPE1 cells (left) and primary cilia length in parental or sgTFEB RPE1 cells treated with DMSO, Torin1, or bafilomycin A1 (right). (L) Immunoblot of parental, sgTSC2, and human TSC2-rescued sgTSC2 RPE1 cells treated with bafilomycin A1 for the final 3 h under overnight serum starvation. (M) Primary cilia length in RPE1 cells used in (L) following treatment with DMSO or bafilomycin A1. All treatments were applied during the final 24 h in serum-free conditions (48 h total), unless otherwise specified: DMSO (0.1%), rapamycin (20 nM), Torin1 (250 nM), chloroquine (50 μM), bafilomycin A1 (100 nM), SBP-7455 (10 μM), and VPS34-IN1 (10 μM). Data are represented as mean ± standard deviation (SD). Statistical analysis was performed using two-way ANOVA followed by Šídák’s multiple comparisons test. The significance threshold (α) was set at 0.05, and all p values are reported.

Article Snippet: Flag-tagged human TSC2 (Flag-TSC2) cDNA from pcDNA3 Flag TSC2 (Addgene; 14129) was PCR-amplified using Phusion High-Fidelity DNA Polymerase (Thermo Scientific; F531S) with primers containing sequences homologous to the insertion site in pMXS-IRES-Blast, a retroviral vector (Cell Biolabs, Inc; RTV-106): 5’-CTAGCTAGTTAATTAAatggactacaaagacgatgacgataaagc-3’ 5’-CGCCGGCCCTCGAGtcacacaaactcggtgaagtcctcc-3’ (Uppercase: homologous to pMXS-IRES-Blast; lowercase: homologous to Flag-TSC2).

Techniques: Flow Cytometry, Western Blot, Standard Deviation

Journal: Nucleic acids research

Article Title: Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome.

doi: 10.1093/nar/gkae070

Figure Lengend Snippet: Figure 2. RBM41 interacts with the U12 and U6atac snRNAs in vitro . ( A ) Consensus RNA motifs bound by RBM41 in vitro and matching sequences in the U12 and U6atac snRNAs. The consensus motif (obtained from ENCODE database ( 81 ), entry ENCSR637HFY) determined by Ray et al. ( 58 ) using the RNAcompete method is shown. ( B ) RNA hairpins used in EMSA experiments and their location in the U12 and U6atac snRNAs. ( C ) EMSA analysis of RBM41 and U11 / U12-65K RRM binding to U12 (top panel) and U6atac snRNA (bottom panel) hairpins. EMSA was carried out using recombinant RBM41 RRM (residues 267–413) or 65K C-terminal RRM (residues 380–517) and 32 P-labeled U12, U6atac or negative control RNA hairpins shown in panel B. ( D ) Binding curves and dissociation constants for the interaction of RBM41 and 65K RRMs with U12 and U6atac hairpins. The inset shows a low protein concentration range (0–10 μM) of the same binding curves.

Article Snippet: For BioID cell line construction, full-length RBM41 (1–413), RBM41 N-terminal fragment (1–258), RBM41 Cterminal fragment (259–413) and full-length 65K were cloned into MAC-tag-N vector (Addgene #108078) using Gateway cloning as described ( 34 ).

Techniques: In Vitro, Binding Assay, Recombinant, Labeling, Negative Control, Protein Concentration

Journal: Nucleic acids research

Article Title: Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome.

doi: 10.1093/nar/gkae070

Figure Lengend Snippet: Figure 3. RBM41 specifically associates with minor spliceosomal snRNPs. ( A ) RNA immunoprecipitation with V5-tagged RBM41 and 65K. V5-RBM41 or V5-65K expression vector or empty vector were transfected into HEK293 cells. 24 h later, RNA immunoprecipitation with anti-V5 antibody or control antibody was carried out in native conditions and co-immunoprecipitated RNA analyzed by northern blot using the indicated probes. ( B ) RNA immunoprecipitation with endogenous RBM41. RIP was carried out in native conditions in either HeLa nuclear extract (left) or HEK293 total lysate (right) using an antibody against endogenous RBM41 or control antibody. ( C ) V5-RBM41 constructs used for RNA immunoprecipitation in panel D. ( D ) Effect of truncations and RRM mutations on the snRNP association of RBM41.V5-tagged RBM41 constructs shown in C were transfected into HEK293 cells and RNA immunoprecipitation carried out using anti-V5 or control antibody.

Article Snippet: For BioID cell line construction, full-length RBM41 (1–413), RBM41 N-terminal fragment (1–258), RBM41 Cterminal fragment (259–413) and full-length 65K were cloned into MAC-tag-N vector (Addgene #108078) using Gateway cloning as described ( 34 ).

Techniques: RNA Immunoprecipitation, Expressing, Plasmid Preparation, Transfection, Control, Immunoprecipitation, Northern Blot, Construct

Journal: Nucleic acids research

Article Title: Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome.

doi: 10.1093/nar/gkae070

Figure Lengend Snippet: Figure 4. RBM41 and U11 / U12-65K partition into distinct snRNP comple x es. ( A ) Glycerol gradient analysis of RBM41 and U11 / U12-65K in HeLa nuclear extract. Nuclear extract was loaded on top of a 10–30% glycerol gradient. After ultracentrifugation, the gradient was fractionated, protein and RNA isolated and analyzed by western and northern blot using the antibodies and probes indicated on the left. Location of the U11, U12 and U6atac mono-snRNPs, U11 / U12 di-snRNP and U4at ac / U6at ac di-snRNP are inferred based on the snRNA profiles. ( B ) Domain str uct ures of MAC-tagged RBM41 and 65K constructs used for BioID. N-terminal MAC tag is not drawn to scale. ( C ) Spectral count fold changes for U11 / U12 di-snRNP proteins in BioID datasets. ( D ) Immunoprecipitation of U11 and U12 snRNAs by anti-31K, anti-48K, anti-59K, anti-65K and anti-Sm antibodies in HEK293 total lysate f ollo w ed b y Northern blot detection of the U11 and U12 snRNAs.

Article Snippet: For BioID cell line construction, full-length RBM41 (1–413), RBM41 N-terminal fragment (1–258), RBM41 Cterminal fragment (259–413) and full-length 65K were cloned into MAC-tag-N vector (Addgene #108078) using Gateway cloning as described ( 34 ).

Techniques: Isolation, Western Blot, Northern Blot, Construct, Immunoprecipitation

Journal: Nucleic acids research

Article Title: Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome.

doi: 10.1093/nar/gkae070

Figure Lengend Snippet: Figure 5. RBM41 interacts with DHX8 and localizes to Cajal bodies. ( A ) Spectral counts for DHX8 in RBM41 and U11 / U12-65K BioID datasets. ( B ) Immunoprecipitation with anti-V5 or control antibody f ollo w ed b y w estern blot in Flp-In™T-REx™293 cell lines e xpressing V5-RBM41 or V5-65K. T he asterisk indicates a non-specific band detected in both control and anti-31K IPs and likely represents cross-reaction of the anti-rabbit secondary antibody with light chain from the IP antibody. ( C ) RNA immunoprecipitation with e x ogenously e xpressed V5-tagged proteins f ollo w ed b y R T-PCR. T he indicated pCI-neo constructs for expressing V5-tagged proteins or empty pCI-neo vectors were transfected into HEK293 cells. 24 h later, RIP was carried out using anti-V5 antibody and RNA extracted from the beads analyzed by RT-PCR. Amplification across the branch junction was used to detect U2- and U12-type intron lariats and lariat intermediates from the f ollo wing introns: SPCS2 introns 3–4 (U12) and 2–3 (U2), SUDS3 introns 7–8 (U12) and 9–10 (U2), WDR11 introns 28–29 (U12) and 27–28 (U2). ( D ) RNA immunoprecipitation with endogenous RBM41 in HEK293 cells f ollo w ed b y R T-PCR. ( E ) Spectral counts for coilin in RBM41 and U11 / U12-65K BioID datasets. ( F ) Anti-RBM41 immunofluorescence in HEK293 cells transfected with a vector f or e xpressing coilin-GFP.

Article Snippet: For BioID cell line construction, full-length RBM41 (1–413), RBM41 N-terminal fragment (1–258), RBM41 Cterminal fragment (259–413) and full-length 65K were cloned into MAC-tag-N vector (Addgene #108078) using Gateway cloning as described ( 34 ).

Techniques: Immunoprecipitation, Control, RNA Immunoprecipitation, Construct, Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Amplification, Immunofluorescence, Plasmid Preparation

Journal: Nucleic acids research

Article Title: Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome.

doi: 10.1093/nar/gkae070

Figure Lengend Snippet: Figure 6. RBM41 knockout influences the splicing of U12-type introns. ( A ) Western blot analysis of RBM41 knockout and matching control cell lines used in the RNAseq analysis. ( B ) Comparison of the statistically significant (Whippet Probability > 0.9) alternative splicing events in the genes containing only U2-type introns and e v ents either within or near proximity (immediate up- or downstream exons and introns) of the U12-type introns. AA - alternative acceptor, AD – alternative donor, CE – core e x on. ( C ) R epresentativ e sashimi plots showing Intron retention ( NOL11 ), Alternative U12-type 3 ′ ss choice ( THOC2 ) and loss of both exon skipping and alternative U12-type 3 ′ ss usage in RBM41 knockout cells ( TCTN1 ). The percentages refer to the intron retention le v els ( NOL11 ), the alternative 3 ′ splice usage le v els ( TH OC2 ) or e x on skipping le v els ( TCTN1 ) as indicated by the arches in the Sashimi plot. ( D ) Validation of the THOC2 and TCTN1 alternative splicing changes using a set of three independent RBM41 knockout cell lines and their matching controls.

Article Snippet: For BioID cell line construction, full-length RBM41 (1–413), RBM41 N-terminal fragment (1–258), RBM41 Cterminal fragment (259–413) and full-length 65K were cloned into MAC-tag-N vector (Addgene #108078) using Gateway cloning as described ( 34 ).

Techniques: Knock-Out, Western Blot, Control, Comparison, Alternative Splicing, Biomarker Discovery