c terminal mbp tagged protein  (Addgene inc)

Journal: bioRxiv

Article Title: Structure of the human outer kinetochore KMN network complex

doi: 10.1101/2023.08.07.552234

Figure Lengend Snippet: a. Molecular model of the Dsn1 N fitted into the MIS12C Head- cryo-EM density map, showing side-chain resolution at the key interaction regions. b. MIS12C:CENP-C structure (grey, PDB ID: 5LSK ) overlayed with the auto-inhibited MIS12C complex shows that the back side of the MIS12C is available to bind a portion of the CENP-C (residues 1-21). c. CENP-C multiple sequence alignment , highlighting the CENP-C regions that can bind to the MIS12C complex in the auto-inhibited state (accessible region, C-terminal portion) and the region of CENP-C (N-terminal portion) that is blocked from engaging MIS12C.

Article Snippet: Synthetic genes for H. sapiens Knl1 2131–2337 and Cenp-C 1–71 , fused to a C-terminal Maltose-Binding Protein (MBP) tag (with the aacgccgccagcggt linker in between), were supplied by Integrated DNA Technologies.

Techniques: Cryo-EM Sample Prep, Sequencing

Journal: bioRxiv

Article Title: Structure of the human outer kinetochore KMN network complex

doi: 10.1101/2023.08.07.552234

Figure Lengend Snippet: a. Coomassie blue-stained SDS-PAGE gels of the MIS12C:CENP-C 1–71 interaction reconstitution. Wild-type MIS12C, MIS12 Dsn1ΔN , MIS12C with W96A/R97A/R98A substitutions in Dsn1 (MIS12C Dsn W96A/R97A/R98A ) or MIS12C Dsn1 S100D/S109D was used in these experiments to test interaction with CENP-C 1–71 tagged at the C-terminus with Maltose Binding Protein (MBP, CENP-C 1–71 -MBP) to allow robust visualization of CENP-C. b. SEC elution chromatograms of all of the experiments described in (a). c. AlphaFold2 Multimer prediction of the K. lactis MIS12C complex structure. Coloured by pLDDT score. d. AlphaFold2 Multimer prediction of the K. lactis MIS12C complex coloured by subunit with insert highlighting conservation of the auto-inhibitory Dsn1 N mechanism.

Article Snippet: Synthetic genes for H. sapiens Knl1 2131–2337 and Cenp-C 1–71 , fused to a C-terminal Maltose-Binding Protein (MBP) tag (with the aacgccgccagcggt linker in between), were supplied by Integrated DNA Technologies.

Techniques: Staining, SDS Page, Binding Assay

Journal: bioRxiv

Article Title: Structure of the human outer kinetochore KMN network complex

doi: 10.1101/2023.08.07.552234

Figure Lengend Snippet: a. Overview of the MIS12C:KNL1C interface. The pair of RWD domains of Knl1 dominate this interface. b. Molecular details of the Knl1 RWD-C interaction with MIS12C stalk, formed by Trp2249 docking into shallow grooves of the MIS12C central stalk and additionally supported by a number of electrostatic interactions on the opposite end of the Knl1 RWD-C . c. The Nsl1 C-terminal peptide augments the central Knl1 RWD-C β-sheet interaction with MIS12C, forming additional contacts with Knl1 RWD-N .

Article Snippet: Synthetic genes for H. sapiens Knl1 2131–2337 and Cenp-C 1–71 , fused to a C-terminal Maltose-Binding Protein (MBP) tag (with the aacgccgccagcggt linker in between), were supplied by Integrated DNA Technologies.

Techniques:

Journal: The EMBO Journal

Article Title: Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning and reduces neurodegeneration

doi: 10.15252/embj.2020105001

Figure Lengend Snippet: hnRNPA2 LC (AlexaFluor 488‐tagged, green) undergoes LLPS, while hnRNPF PLD (AlexaFluor 555‐tagged, red) does not. However, hnRNPF PLD partitions into hnRNPA2 LC droplets when mixed at a 1:1 ratio. Conditions: 20 µM indicated protein (~1% fluorescently tagged), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 10 µm. At 300 µM, FUS LC (AlexaFluor488‐tagged, green) undergoes LLPS, but at 200 µM hnRNPF PLD (AlexaFluor555‐tagged, red) still does not undergo LLPS. When mixed at 300 µM FUS LC and 200 µM hnRNPF PLD, hnRNPF PLD does not partition into FUS LC droplets. Conditions: 300 µM FUS and 200 µM hnRNPF PLD (~1% fluorescently tagged), 20 mM MES pH 5.5, 150 mM NaCl, 150 mM urea. Scale bar: 10 µm. While FUS LC and hnRNPF PLD both have a small negative predicted net charge at neutral pH, hnRNPA2 LC has a predicted + 4 net positive charge, due to the 9 positively charged residues (8 arginine, 1 lysine) and 5 negatively charged residues. Removal of the charged residues from hnRNPA2 LC (hnRNPA2 LC CD ) prevents partitioning of hnRNPF PLD into the hnRNPA2 LC phase. Addition of hnRNPA2 LC‐like charged residue patterning to FUS LC (FUS LC CE ) allows the partitioning of hnRNPF PLD at 40 µM. Conditions: protein concentration indicated next to image (20 µM hnRNPA2 LC and hnRNPA2 LC CD , 40 µM FUS LC CE , hnRNPF PLD concentration matches other protein in mixture (either 20 or 40 µM)) (all ~ 1% fluorescently tagged), 20 mM MES pH 5.5 50 mM NaCl, 150 mM urea. Scale bar: 10 µm. Substitution of all arginines in hnRNPA2 LC with lysine prevents the partitioning of hnRNPF PLD into hnRNPA2 LC R→K droplets. Removing all charged residues except for arginine from hnRNPA2 LC (hnRNPA2 LC CD,R ) allows partitioning of hnRNPF PLD into droplets, indicating arginine in hnRNPA2 LC is required and necessary for hnRNPF partitioning. hnRNPA2 LC R→K does not phase separate much as hnRNPA2 LC at these conditions, see Appendix Fig for quantification of phase separation of variants. Conditions: 20 µM proteins, 20 mM MES pH 5.5 50 mM NaCl, 150 mM urea. Scale bar: 10 µm. Quantification of phase separation of hnRNPA2 LC constructs used to determine the residue types important for hnRNPF PLD partitioning. hnRNPA2 LC N→S (purple) has similar phase separation to hnRNPA2 LC. hnRNPA2 LC CD (red) is consistently phase separated with ~ 5 µM protein remaining in the supernatant at all salt conditions tested. Adding back arginines to hnRNPA2 LC no charge (hnRNPA2 LC CD,R , green) brings phase separation as a function of salt to similar levels as hnRNPA2 LC. Changing all the arginine residues to lysine (removing the π‐character but maintaining positive charge, hnRNPA2 LC R→K ) also removes the salt dependence of phase separation but has reduced phase separation overall. Conditions: 20 µM of each protein, pH 5.5 MES, NaCl concentration as indicated, 25° C. Error bars are standard deviation of three replicates.

Article Snippet: The following constructs and general purification strategies were used for protein expression in BL21 Star (DE3) E. coli cultures (Life Technologies): hnRNPA2 LC (190–341), insoluble His‐tag purification as described (Ryan et al , ) (Addgene ID: 98657) hnRNPA2 LC S285C and S329C variants for PRE, insoluble His‐tag purification as described (Ryan et al , ) (Addgene ID: 98665, 98667, respectively) MBP‐hnRNPA2 LC, soluble His‐tag purification as described (Ryan et al , ) (Addgene ID: 98661) FUS LC and FUS LC 12E, soluble His‐tag purifications as described (Monahan et al , ) (Addgene ID: 98653, 98654) C‐terminal maltose‐binding protein‐tagged hnRNPA2 FL WT, D290V, and P298L, soluble His‐tag purification (Addgene ID: 139109, 139110, 139111, respectively) TOG D1, soluble His‐tag purification (Addgene ID: 139112) hnRNPF PLD, His‐tag purification (Addgene ID: 139113) N‐terminal maltose‐binding protein‐tagged hnRNPF FL and hnRNPF ∆PLD, soluble His‐tag purification (Addgene ID: 139114, 139115, respectively) HRPA‐1 LC, insoluble His‐tag purification (Addgene ID: 139116) C‐terminal maltose‐binding protein‐tagged HRPA‐1 FL, soluble His‐tag purification (Addgene ID: 139117) hnRNPA2 LC CD , hnRNPA2 LC CD,R , hnRNPA2 LC R→K , hnRNPA2 N→S , hnRNPA2 LC 5E , hnRNPA2 LC 12E , insoluble His‐tag purification (Addgene ID: 139118, 139119, 139120, 139121, 139122, 139123, respectively) hnRNPF PLD Y→S , hnRNPF PLD S→A , His‐tag purification (Addgene ID: 139124, 139125, respectively) FUS LC CE , FUS LC CE,R→K , FUS LC R , insoluble His‐tag purification (Addgene ID: 139126, 139127, 139128)

Techniques: Residue, Protein Concentration, Concentration Assay, Construct, Standard Deviation

Journal: The EMBO Journal

Article Title: Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning and reduces neurodegeneration

doi: 10.15252/embj.2020105001

Figure Lengend Snippet: A AlexaFluor 488‐tagged (green) hnRNPA2 LC undergoes LLPS, while AlexaFluor 555‐tagged (red) TOG D1 does not. However, TOG D1 partitions into hnRNPA2 LC droplets when mixed at a 1:1 ratio. Conditions: 20 µM indicated protein (~1% fluorescently tagged), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 10 µm. hnRNPA2 LC control duplicated from Fig as hnRNPF PLD and TOG D1 samples were made concurrently. B Similar to hnRNPF PLD, AlexaFluor 555‐tagged TOG D1 does not undergo LLPS at 300 µM or partition into AlexaFluor488‐tagged FUS LC droplets with both proteins at 300 µM. Conditions: 300 µM proteins (~1% fluorescently tagged), 20 mM MES pH 5.5 150 mM NaCl, 150 mM urea. Scale bar: 10 µm. C, D TOG D1 homology structure with Γ 2 values from PRE experiments for hnRNPA2 LC (C) S285C and (D) S329C. Amino acids are colored based on Γ 2 value: Red corresponds to Γ 2 > 2, orange to 2 > Γ 2 > 1, yellow to 1 > Γ 2 > 0.5. E AlexaFluor 555‐tagged hnRNPF PLD or FL and AlexaFluor 405‐tagged TOG D1 partition simultaneously into AlexaFluor 488‐tagged hnRNPA2 LC droplets. Conditions: 20 µM of each indicated protein (~1% fluorescently tagged), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 20 µm.

Article Snippet: The following constructs and general purification strategies were used for protein expression in BL21 Star (DE3) E. coli cultures (Life Technologies): hnRNPA2 LC (190–341), insoluble His‐tag purification as described (Ryan et al , ) (Addgene ID: 98657) hnRNPA2 LC S285C and S329C variants for PRE, insoluble His‐tag purification as described (Ryan et al , ) (Addgene ID: 98665, 98667, respectively) MBP‐hnRNPA2 LC, soluble His‐tag purification as described (Ryan et al , ) (Addgene ID: 98661) FUS LC and FUS LC 12E, soluble His‐tag purifications as described (Monahan et al , ) (Addgene ID: 98653, 98654) C‐terminal maltose‐binding protein‐tagged hnRNPA2 FL WT, D290V, and P298L, soluble His‐tag purification (Addgene ID: 139109, 139110, 139111, respectively) TOG D1, soluble His‐tag purification (Addgene ID: 139112) hnRNPF PLD, His‐tag purification (Addgene ID: 139113) N‐terminal maltose‐binding protein‐tagged hnRNPF FL and hnRNPF ∆PLD, soluble His‐tag purification (Addgene ID: 139114, 139115, respectively) HRPA‐1 LC, insoluble His‐tag purification (Addgene ID: 139116) C‐terminal maltose‐binding protein‐tagged HRPA‐1 FL, soluble His‐tag purification (Addgene ID: 139117) hnRNPA2 LC CD , hnRNPA2 LC CD,R , hnRNPA2 LC R→K , hnRNPA2 N→S , hnRNPA2 LC 5E , hnRNPA2 LC 12E , insoluble His‐tag purification (Addgene ID: 139118, 139119, 139120, 139121, 139122, 139123, respectively) hnRNPF PLD Y→S , hnRNPF PLD S→A , His‐tag purification (Addgene ID: 139124, 139125, respectively) FUS LC CE , FUS LC CE,R→K , FUS LC R , insoluble His‐tag purification (Addgene ID: 139126, 139127, 139128)

Techniques: Control

Journal: The EMBO Journal

Article Title: Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning and reduces neurodegeneration

doi: 10.15252/embj.2020105001

Figure Lengend Snippet: Concentration of protein remaining in the supernatant after centrifugation, which is an inverse measure of phase separation, changes with salt concentration. hnRNPA2 LC shows low phase separation (high protein remaining in the supernatant) at low salt conditions and near‐complete phase separation (no protein remaining in the supernatant) at high salt concentrations. In contrast, tyrosine phosphorylated hnRNPA2 LC shows higher phase separation (less protein remaining in the supernatant) at low salt concentrations than at high salt concentrations. Conditions: 20 µM protein, 20 mM MES pH 5.5 150 mM urea, salt concentration as indicated, 25°C. Error bars are standard deviation of three replicates. While hnRNPA2 LC shows no droplets in low salt conditions and droplets in high salt conditions, tyrosine phosphorylated hnRNPA2 LC shows droplets in all salt concentrations tested, although more droplets are present at low salt conditions. Conditions: 20 µM proteins, 20 mM MES pH 5.5 150 mM urea with 0 mM, 50 mM, or 500 mM NaCl as indicated. Scale bar: 10 µm. Fluorescence micrographs showing that although TOG D1 and hnRNPF PLD partition into hnRNPA2 LC droplets (rows 1 and 3), they are unable to partition into tyrosine phosphorylated hnRNPA2 LC droplets (rows 2 and 4). Conditions: 20 µM proteins (~1% fluorescently labeled), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 10 µm. Phosphomimic variants containing 5 or 12 serine to glutamate substitutions (hnRNPA2 LC 5E and hnRNPA2 LC 12E , respectively) both allow partitioning of TOG D1 and hnRNPF PLD, even though hnRNPA2 LC 12E barely phase separates at these conditions, indicating that increased negative charge is insufficient to prevent partitioning of TOG D1 and hnRNPF PLD. See Appendix Fig for quantification of LLPS of hnRNPA2 LC 5E and hnRNPA2 LC 12E . Conditions: 20 µM proteins (~1% fluorescently labeled), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 10 µm.

Article Snippet: The following constructs and general purification strategies were used for protein expression in BL21 Star (DE3) E. coli cultures (Life Technologies): hnRNPA2 LC (190–341), insoluble His‐tag purification as described (Ryan et al , ) (Addgene ID: 98657) hnRNPA2 LC S285C and S329C variants for PRE, insoluble His‐tag purification as described (Ryan et al , ) (Addgene ID: 98665, 98667, respectively) MBP‐hnRNPA2 LC, soluble His‐tag purification as described (Ryan et al , ) (Addgene ID: 98661) FUS LC and FUS LC 12E, soluble His‐tag purifications as described (Monahan et al , ) (Addgene ID: 98653, 98654) C‐terminal maltose‐binding protein‐tagged hnRNPA2 FL WT, D290V, and P298L, soluble His‐tag purification (Addgene ID: 139109, 139110, 139111, respectively) TOG D1, soluble His‐tag purification (Addgene ID: 139112) hnRNPF PLD, His‐tag purification (Addgene ID: 139113) N‐terminal maltose‐binding protein‐tagged hnRNPF FL and hnRNPF ∆PLD, soluble His‐tag purification (Addgene ID: 139114, 139115, respectively) HRPA‐1 LC, insoluble His‐tag purification (Addgene ID: 139116) C‐terminal maltose‐binding protein‐tagged HRPA‐1 FL, soluble His‐tag purification (Addgene ID: 139117) hnRNPA2 LC CD , hnRNPA2 LC CD,R , hnRNPA2 LC R→K , hnRNPA2 N→S , hnRNPA2 LC 5E , hnRNPA2 LC 12E , insoluble His‐tag purification (Addgene ID: 139118, 139119, 139120, 139121, 139122, 139123, respectively) hnRNPF PLD Y→S , hnRNPF PLD S→A , His‐tag purification (Addgene ID: 139124, 139125, respectively) FUS LC CE , FUS LC CE,R→K , FUS LC R , insoluble His‐tag purification (Addgene ID: 139126, 139127, 139128)

Techniques: Concentration Assay, Centrifugation, Standard Deviation, Fluorescence, Labeling

Journal: The EMBO Journal

Article Title: Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning and reduces neurodegeneration

doi: 10.15252/embj.2020105001

Figure Lengend Snippet: After cleavage of a C‐terminal maltose‐binding protein solubility tag, hnRNPA2 FL WT undergoes LLPS and disease mutants D290V and P298L aggregate. In contrast, phosphorylated hnRNPA2 FL WT and P298L do not undergo LLPS or aggregation in the time frame tested. Phosphorylated hnRNPA2 FL D290V can form some structures resembling liquid droplets but does not form amorphous aggregates in the time frame tested. Conditions: 20 µM proteins, 20 mM Tris pH 7.4, 50 mM NaCl. Scale bar: 10 µm. PLAAC analysis (Lancaster et al , ) of hnRNPA2 (black), changing all tyrosine residues in hnRNPA2 LC to glutamate (17 Y→E, red), 5 serine to glutamate phosphomimic mutations (5 S→E, blue), all serine to glutamate (12 S→E, purple), and all tyrosine to glutamine (17 Y→Q) shows that hnRNPA2 LC prion‐like character reduces slightly with serine phosphomimic mutations, but decreases drastically with tyrosine phosphomimic mutations. As the number of phosphorylation events increases, the radius of gyration ( R g ) of hnRNPA2 LC increases in coarse‐grained simulations. R g uncertainty bars represent S.E.M. from 10 different simulations with the same number of phosphorylated tyrosines randomly placed in different positions. Coarse‐grained simulations of WT show that compared to the unphosphorylated form, phosphotyrosine hnRNPA2 LC forms fewer contacts with itself.

Article Snippet: The following constructs and general purification strategies were used for protein expression in BL21 Star (DE3) E. coli cultures (Life Technologies): hnRNPA2 LC (190–341), insoluble His‐tag purification as described (Ryan et al , ) (Addgene ID: 98657) hnRNPA2 LC S285C and S329C variants for PRE, insoluble His‐tag purification as described (Ryan et al , ) (Addgene ID: 98665, 98667, respectively) MBP‐hnRNPA2 LC, soluble His‐tag purification as described (Ryan et al , ) (Addgene ID: 98661) FUS LC and FUS LC 12E, soluble His‐tag purifications as described (Monahan et al , ) (Addgene ID: 98653, 98654) C‐terminal maltose‐binding protein‐tagged hnRNPA2 FL WT, D290V, and P298L, soluble His‐tag purification (Addgene ID: 139109, 139110, 139111, respectively) TOG D1, soluble His‐tag purification (Addgene ID: 139112) hnRNPF PLD, His‐tag purification (Addgene ID: 139113) N‐terminal maltose‐binding protein‐tagged hnRNPF FL and hnRNPF ∆PLD, soluble His‐tag purification (Addgene ID: 139114, 139115, respectively) HRPA‐1 LC, insoluble His‐tag purification (Addgene ID: 139116) C‐terminal maltose‐binding protein‐tagged HRPA‐1 FL, soluble His‐tag purification (Addgene ID: 139117) hnRNPA2 LC CD , hnRNPA2 LC CD,R , hnRNPA2 LC R→K , hnRNPA2 N→S , hnRNPA2 LC 5E , hnRNPA2 LC 12E , insoluble His‐tag purification (Addgene ID: 139118, 139119, 139120, 139121, 139122, 139123, respectively) hnRNPF PLD Y→S , hnRNPF PLD S→A , His‐tag purification (Addgene ID: 139124, 139125, respectively) FUS LC CE , FUS LC CE,R→K , FUS LC R , insoluble His‐tag purification (Addgene ID: 139126, 139127, 139128)

Techniques: Binding Assay, Solubility, Phospho-proteomics

Journal: The EMBO Journal

Article Title: Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning and reduces neurodegeneration

doi: 10.15252/embj.2020105001

Figure Lengend Snippet: hnRNPA2 interacts with transport granule components hnRNPF and ch‐TOG in the phase‐separated state. Disease mutations (X) D290V and P298L induce aggregation of hnRNPA2 in vitro , while D290V induces tdp‐1 ‐dependent neurodegeneration in a C. elegans model. hnRNPA2 LC tyrosine phosphorylation alters hnRNPA2 LC LLPS in vitro , prevents interaction with hnRNPF and ch‐TOG, reduces aggregation in vitro , and expression of an activated tyrosine kinase reduces D290V‐associated neurodegeneration in the C. elegans model.

Article Snippet: The following constructs and general purification strategies were used for protein expression in BL21 Star (DE3) E. coli cultures (Life Technologies): hnRNPA2 LC (190–341), insoluble His‐tag purification as described (Ryan et al , ) (Addgene ID: 98657) hnRNPA2 LC S285C and S329C variants for PRE, insoluble His‐tag purification as described (Ryan et al , ) (Addgene ID: 98665, 98667, respectively) MBP‐hnRNPA2 LC, soluble His‐tag purification as described (Ryan et al , ) (Addgene ID: 98661) FUS LC and FUS LC 12E, soluble His‐tag purifications as described (Monahan et al , ) (Addgene ID: 98653, 98654) C‐terminal maltose‐binding protein‐tagged hnRNPA2 FL WT, D290V, and P298L, soluble His‐tag purification (Addgene ID: 139109, 139110, 139111, respectively) TOG D1, soluble His‐tag purification (Addgene ID: 139112) hnRNPF PLD, His‐tag purification (Addgene ID: 139113) N‐terminal maltose‐binding protein‐tagged hnRNPF FL and hnRNPF ∆PLD, soluble His‐tag purification (Addgene ID: 139114, 139115, respectively) HRPA‐1 LC, insoluble His‐tag purification (Addgene ID: 139116) C‐terminal maltose‐binding protein‐tagged HRPA‐1 FL, soluble His‐tag purification (Addgene ID: 139117) hnRNPA2 LC CD , hnRNPA2 LC CD,R , hnRNPA2 LC R→K , hnRNPA2 N→S , hnRNPA2 LC 5E , hnRNPA2 LC 12E , insoluble His‐tag purification (Addgene ID: 139118, 139119, 139120, 139121, 139122, 139123, respectively) hnRNPF PLD Y→S , hnRNPF PLD S→A , His‐tag purification (Addgene ID: 139124, 139125, respectively) FUS LC CE , FUS LC CE,R→K , FUS LC R , insoluble His‐tag purification (Addgene ID: 139126, 139127, 139128)

Techniques: In Vitro, Phospho-proteomics, Expressing

Journal: The EMBO Journal

Article Title: Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning and reduces neurodegeneration

doi: 10.15252/embj.2020105001

Figure Lengend Snippet:

Article Snippet: The following constructs and general purification strategies were used for protein expression in BL21 Star (DE3) E. coli cultures (Life Technologies): hnRNPA2 LC (190–341), insoluble His‐tag purification as described (Ryan et al , ) (Addgene ID: 98657) hnRNPA2 LC S285C and S329C variants for PRE, insoluble His‐tag purification as described (Ryan et al , ) (Addgene ID: 98665, 98667, respectively) MBP‐hnRNPA2 LC, soluble His‐tag purification as described (Ryan et al , ) (Addgene ID: 98661) FUS LC and FUS LC 12E, soluble His‐tag purifications as described (Monahan et al , ) (Addgene ID: 98653, 98654) C‐terminal maltose‐binding protein‐tagged hnRNPA2 FL WT, D290V, and P298L, soluble His‐tag purification (Addgene ID: 139109, 139110, 139111, respectively) TOG D1, soluble His‐tag purification (Addgene ID: 139112) hnRNPF PLD, His‐tag purification (Addgene ID: 139113) N‐terminal maltose‐binding protein‐tagged hnRNPF FL and hnRNPF ∆PLD, soluble His‐tag purification (Addgene ID: 139114, 139115, respectively) HRPA‐1 LC, insoluble His‐tag purification (Addgene ID: 139116) C‐terminal maltose‐binding protein‐tagged HRPA‐1 FL, soluble His‐tag purification (Addgene ID: 139117) hnRNPA2 LC CD , hnRNPA2 LC CD,R , hnRNPA2 LC R→K , hnRNPA2 N→S , hnRNPA2 LC 5E , hnRNPA2 LC 12E , insoluble His‐tag purification (Addgene ID: 139118, 139119, 139120, 139121, 139122, 139123, respectively) hnRNPF PLD Y→S , hnRNPF PLD S→A , His‐tag purification (Addgene ID: 139124, 139125, respectively) FUS LC CE , FUS LC CE,R→K , FUS LC R , insoluble His‐tag purification (Addgene ID: 139126, 139127, 139128)

Techniques: