destination vector pdest 2xflag  (Addgene inc)

Journal: bioRxiv

Article Title: Structural basis for the midnolin-proteasome pathway and its role in suppressing myeloma

doi: 10.1101/2025.02.22.639686

Figure Lengend Snippet: (A) Midnolin contains three domains necessary for its degradative function: a ubiquitin-like (Ubl) domain, the Catch domain, and a C-terminal helix (αHelix-C) containing a nuclear localization sequence. (B) Schematic representation of the process to affinity-purify the midnolin-proteasome complex by size-exclusion chromatography from HEK-293T cells transiently overexpressing 2xFLAG-tagged midnolin variants. Created with BioRender.com. (C) A representative size-exclusion chromatography trace indicating the fractions collected corresponding to the midnolin-proteasome complex. (D) A representative Coomassie stain of the purified midnolin-proteasome complex showing the characteristic migration pattern of the proteasomal subunits after SDS-PAGE. (E) A representative negative stain electron micrograph of the midnolin-proteasome complex.

Article Snippet: Wild type and mutant versions of entry clones were subcloned into the following destination vectors via an LR reaction (Thermo Fisher Scientific, 11791100): pHAGE CMV 2xFLAG-destination vector for N-terminally tagging MIDN, IRF4, NeuroD1, and SPINDOC, a pHAGE CMV 2xHA-destination vector for N-terminally tagging PSMD2, a CMV-C-2xFLAG destination vector (Addgene, 118372) for C-terminally tagging EGR1, a pHAGE CMV 2xFLAG-MBP-destination vector for N-terminally tagging the midnolin α-HelixC, a GPS 3.0 destination vector for GFP-IRF4, a GPS 3.2 destination vector for EGR1-GFP, or a pHAGE EF1α-destination vector (blue fluorescent protein, BFP) for expressing untagged midnolin for flow cytometry experiments.

Techniques: Sequencing, Size-exclusion Chromatography, Staining, Purification, Migration, SDS Page

Journal: bioRxiv

Article Title: Structural basis for the midnolin-proteasome pathway and its role in suppressing myeloma

doi: 10.1101/2025.02.22.639686

Figure Lengend Snippet: (A) Atomic model of midnolin αHelix-C in complex with PSMD2/Rpn1, highlighting the role of the midnolin nuclear localization sequence (NLS) in mediating the interaction. (B) Immunoblotting of anti-FLAG immunoprecipitants from MIDN knockout HEK-293T cells transiently overexpressing 2xFLAG-midnolin using a CMV promoter. (C) Anti-FLAG immunofluorescence of MIDN knockout HEK-293T cells stably expressing 2xFLAG-midnolin. Cells were treated with 10 µM MG132 for 4 hours. (D) In vitro co-immunoprecipitation followed by immunoblotting. Purified FLAG-MsyB-αHelix-C were immobilized onto anti-FLAG beads and incubated with pure TNPO1 or KPNA2, (E) human proteasomes, or (F) human proteasomes with either TNPO1 or KPNA2.

Article Snippet: Wild type and mutant versions of entry clones were subcloned into the following destination vectors via an LR reaction (Thermo Fisher Scientific, 11791100): pHAGE CMV 2xFLAG-destination vector for N-terminally tagging MIDN, IRF4, NeuroD1, and SPINDOC, a pHAGE CMV 2xHA-destination vector for N-terminally tagging PSMD2, a CMV-C-2xFLAG destination vector (Addgene, 118372) for C-terminally tagging EGR1, a pHAGE CMV 2xFLAG-MBP-destination vector for N-terminally tagging the midnolin α-HelixC, a GPS 3.0 destination vector for GFP-IRF4, a GPS 3.2 destination vector for EGR1-GFP, or a pHAGE EF1α-destination vector (blue fluorescent protein, BFP) for expressing untagged midnolin for flow cytometry experiments.

Techniques: Sequencing, Western Blot, Knock-Out, Immunofluorescence, Stable Transfection, Expressing, In Vitro, Immunoprecipitation, Purification, Incubation

Journal: bioRxiv

Article Title: Structural basis for the midnolin-proteasome pathway and its role in suppressing myeloma

doi: 10.1101/2025.02.22.639686

Figure Lengend Snippet: (A) AlphaFold-multimer prediction of full-length midnolin with PSMD2/Rpn1. The M site represents PSMD2 residues that make direct contact with αHelix-C. The arginine residues within the midnolin nuclear localization sequence (NLS) mediate a part of the interaction. (B) MIDN KO HEK-293T cells were first reconstituted with 2xFLAG-midnolin from a CMV promoter using lentivirus. These cells were then transfected with 2xHA-PSMD2. Shown is immunoblotting from anti-HA immunoprecipitates.

Article Snippet: Wild type and mutant versions of entry clones were subcloned into the following destination vectors via an LR reaction (Thermo Fisher Scientific, 11791100): pHAGE CMV 2xFLAG-destination vector for N-terminally tagging MIDN, IRF4, NeuroD1, and SPINDOC, a pHAGE CMV 2xHA-destination vector for N-terminally tagging PSMD2, a CMV-C-2xFLAG destination vector (Addgene, 118372) for C-terminally tagging EGR1, a pHAGE CMV 2xFLAG-MBP-destination vector for N-terminally tagging the midnolin α-HelixC, a GPS 3.0 destination vector for GFP-IRF4, a GPS 3.2 destination vector for EGR1-GFP, or a pHAGE EF1α-destination vector (blue fluorescent protein, BFP) for expressing untagged midnolin for flow cytometry experiments.

Techniques: Sequencing, Transfection, Western Blot

Journal: bioRxiv

Article Title: Structural basis for the midnolin-proteasome pathway and its role in suppressing myeloma

doi: 10.1101/2025.02.22.639686

Figure Lengend Snippet: (A) Immunoblotting of anti-FLAG immunoprecipitants of MIDN knockout HEK-293T cells transiently overexpressing 2xFLAG-MBP-αHelix-C variants. (B) Anti-FLAG immunofluorescence of MIDN knockout HEK-293T cells stably expressing 2xFLAG-MBP-αHelix-C. (C) Summary of the most enriched co-immunoprecipitated proteins using cell lines from (a) as detected using mass spectrometry. (D) Predicted aligned error (PAE) graph of an (E) AlphaFold3 prediction of full-length midnolin with transportin-1. (F) Immunoblotting was performed from anti-FLAG immunoprecipitants of MIDN KO HEK-293T cells that were transiently overexpressing 2xFLAG-tagged midnolin using a CMV promoter. (G) Same assay as (b) but from MIDN KO HEK-293T cells stably expressing 2xFLAG-midnolin variants. The cells were treated with 10 µM MG132 for 4 hours.

Article Snippet: Wild type and mutant versions of entry clones were subcloned into the following destination vectors via an LR reaction (Thermo Fisher Scientific, 11791100): pHAGE CMV 2xFLAG-destination vector for N-terminally tagging MIDN, IRF4, NeuroD1, and SPINDOC, a pHAGE CMV 2xHA-destination vector for N-terminally tagging PSMD2, a CMV-C-2xFLAG destination vector (Addgene, 118372) for C-terminally tagging EGR1, a pHAGE CMV 2xFLAG-MBP-destination vector for N-terminally tagging the midnolin α-HelixC, a GPS 3.0 destination vector for GFP-IRF4, a GPS 3.2 destination vector for EGR1-GFP, or a pHAGE EF1α-destination vector (blue fluorescent protein, BFP) for expressing untagged midnolin for flow cytometry experiments.

Techniques: Western Blot, Knock-Out, Immunofluorescence, Stable Transfection, Expressing, Immunoprecipitation, Mass Spectrometry

Journal: bioRxiv

Article Title: Structural basis for the midnolin-proteasome pathway and its role in suppressing myeloma

doi: 10.1101/2025.02.22.639686

Figure Lengend Snippet: (A) AlphaFold prediction of a minimal midnolin found in Dimorphilus gyrociliatus (segmented worm) that contains only the three functional domains: Ubl, Catch, and αHelix-C. (B) anti-FLAG immunofluorescence of MIDN KO HEK-293T cells stably expressing 2xFLAG-tagged midnolin variants. Cells were pre-treated with 10 µM MG132 for 4 hours. (C) Immunoblotting from anti-FLAG immunoprecipitations of MIDN KO HEK-293T cells transiently overexpressing 2xFLAG-tagged midnolin variants using a CMV promoter. (D) MIDN KO HEK-293T cells stably expressing a dual-fluorescence EGR1 stability reporter were transfected with control BFP or midnolin co-expressing BFP using an EF-1α promoter. The BFP+ cells (∼10,000) were analyzed for the GFP/DsRed ratio two days post-transfection by flow cytometry.

Article Snippet: Wild type and mutant versions of entry clones were subcloned into the following destination vectors via an LR reaction (Thermo Fisher Scientific, 11791100): pHAGE CMV 2xFLAG-destination vector for N-terminally tagging MIDN, IRF4, NeuroD1, and SPINDOC, a pHAGE CMV 2xHA-destination vector for N-terminally tagging PSMD2, a CMV-C-2xFLAG destination vector (Addgene, 118372) for C-terminally tagging EGR1, a pHAGE CMV 2xFLAG-MBP-destination vector for N-terminally tagging the midnolin α-HelixC, a GPS 3.0 destination vector for GFP-IRF4, a GPS 3.2 destination vector for EGR1-GFP, or a pHAGE EF1α-destination vector (blue fluorescent protein, BFP) for expressing untagged midnolin for flow cytometry experiments.

Techniques: Functional Assay, Immunofluorescence, Stable Transfection, Expressing, Western Blot, Fluorescence, Transfection, Control, Flow Cytometry

Journal: bioRxiv

Article Title: Structural basis for the midnolin-proteasome pathway and its role in suppressing myeloma

doi: 10.1101/2025.02.22.639686

Figure Lengend Snippet: (A) Crystal structure of the EGR1-Catch fusion protein at 2.5 Å resolution. The interaction between EGR1 and the Catch domain is facilitated by alternating phenylalanine-glycine residues, forming an FG zipper. (B) Immunoblotting of anti-FLAG immunoprecipitates from HEK-293T cells expressing endogenous 3xHA-midnolin and transiently overexpressing EGR1-2xFLAG via a CMV promoter. Cells were treated with 10 µM MG132 for 4 hours. (C) AlphaFold-multimer prediction of the IRF4-midnolin complex shows an incomplete FG zipper, with valine 216 replacing tyrosine. The same immunoblot assay as in (b) was performed using cells transfected with 2xFLAG-IRF4. (D) AlphaFold-multimer prediction of the NeuroD1-midnolin interaction reveals an incomplete FG zipper, with isoleucine 279 replacing tyrosine and phenylalanine 285 sterically clashing with phenylalanine 280 of midnolin. The same assay as in (b) was conducted with cells transfected with 2xFLAG-NeuroD1. (E) AlphaFold-multimer prediction of SPINDOC-midnolin shows a missing FG zipper, with glycine 319, leucine 321, and leucine 323 substituting for tyrosine, glycine, and phenylalanine, respectively. The same assay as in (b) with cells transfected with 2xFLAG-SPINDOC. (F) The same assay as in (b) was performed using MIDN knockout HEK-293T cells reconstituted with either wild-type or zipper-swapped 2xHA-midnolin from a CMV promoter using lentivirus.

Article Snippet: Wild type and mutant versions of entry clones were subcloned into the following destination vectors via an LR reaction (Thermo Fisher Scientific, 11791100): pHAGE CMV 2xFLAG-destination vector for N-terminally tagging MIDN, IRF4, NeuroD1, and SPINDOC, a pHAGE CMV 2xHA-destination vector for N-terminally tagging PSMD2, a CMV-C-2xFLAG destination vector (Addgene, 118372) for C-terminally tagging EGR1, a pHAGE CMV 2xFLAG-MBP-destination vector for N-terminally tagging the midnolin α-HelixC, a GPS 3.0 destination vector for GFP-IRF4, a GPS 3.2 destination vector for EGR1-GFP, or a pHAGE EF1α-destination vector (blue fluorescent protein, BFP) for expressing untagged midnolin for flow cytometry experiments.

Techniques: Western Blot, Expressing, Transfection, Knock-Out