anti ranbp9  (Proteintech)

Journal: FEBS letters

Article Title: Skraban-Deardorff intellectual disability syndrome-associated mutations in WDR26 impair CTLH E3 complex assembly.

doi: 10.1002/1873-3468.14866

Figure Lengend Snippet: Fig. 3. WDR26 mutants affect formation of supramolecular WDR26-CTLH E3 assembly. (A) Cartoon of the wildtype supramolecular CTLH E3 assembly and the potential CTLH (sub-) complexes (I, II, and III) caused by WDR26 SKDEAS-associated mutations. Group I: variants maintaining formation of the supramolecular CTLH E3 assembly; group II: variants disrupting CTLH E3 supramolecular assembly but retaining interactions with RANBP9; and group III: variants abolishing both the higher order CTLH E3 assembly and interactions with RANBP9. HA-tagged WDR26 subunits are indicated. (B) Cell lysates of K562 parental, WDR26- and MKLN1-deficient double knockout K562 cells (WDR26/; MKLN1/), and WDR26/; MKLN1/ cells with stably reintroduced HA-tagged WDR26 were fractionated on a continuous 5–40% sucrose gradient, and fractions analyzed by immunoblotting. Fractions with supramolecular assemblies > 670 kDa are indicated with a red box, and smaller subcomplexes with a blue box. (C, D) Cell lysates of K562 WDR26/; MKLN1/ stably reintroduced HA-tagged WDR26 variants from different individuals (Ind#) were fractionated on a continuous 5–40% sucrose gradient, and fractions analyzed by immunoblotting. Supramolecular assemblies and subcomplexes are boxed in red and blue, respectively. Immunoblot data of individual WDR26 variant are grouped into LisH-CTLH-CRA mutants (C) and WDR26 b-propeller mutants (D). Uncropped blots are provided in Figs S1–S3.

Article Snippet: Membranes were incubated over night at 4 °C with the primary antibodies: HA (C29F4) Rabbit mAb (CST, Danvers, MA, USA; 3724S, RRID: AB_1549585, lot 10 + 11, 1 : 2000), RANBP9 Rabbit pAB (Novus Biologicals, Centannial, CO, USA; PAB16671, RRID: AB_10677213, lot 3, 1 : 1000), MAEA Sheep pAb (R&D Systems, Minneapolis, MN, USA; AF7288, RRID: AB_10971438, CGG10119091, 1 : 1000), YPEL5 pAb (Thermo Fisher Scientific; PA5-26957, RRID: AB_2544457, lot VH3047907, 1 : 1000 in 5% BSA, TBS-T), ACTIN (CST; 4967L, RRID: AB_330288, lot 3, 1 : 1000) or DYKDDDDK Tag (D6W5B) Rabbit mAb (CST; 14793S, RRID: AB_2572291, lot 4, 1 : 1000).

Techniques: Double Knockout, Stable Transfection, Western Blot, Variant Assay

Journal: eLife

Article Title: Modular UBE2H-CTLH E2-E3 complexes regulate erythroid maturation

doi: 10.7554/elife.77937

Figure Lengend Snippet: Figure 2. Erythroid maturation stage-dependent modulation of RANBP9- and RANBP10-assembled CTLH complexes. (A) Cartoon of the supramolecular RANBP9-CTLH assembly indicating the catalytic (blue), core (grey/yellow), and supramolecular assembly (orange) modules. (B) Model of remodelling RANBP9- and RANBP10-CTLH complexes. (C) HUDEP2 cell lysates from differentiation days 0 and 6 were separated on sucrose gradients, and fractions analysed by immunoblotting with indicated antibodies. Fractions containing supramolecular CTLH assemblies are boxed in

Article Snippet: For immunoblot analysis, lysates were denatured with SDS sample buffer, boiled at 95°C for 5 min, separated on SDS- PAGE, and proteins were visualized by immunoblotting using indicated primary antibodies: RMND5A (Santa Cruz), MAEA (R&D Systems), RANBP9 (Novus Biologicals), RANBP10 (Invitrogen, #PA5- 110267), TWA1 (Thermo Fisher), ARMC8 (Santa Cruz), WDR26 (Bethyl Laboratories), MKLN1 (Santa Cruz), YPEL5 (Thermo Fisher), GID4 (described in Sherpa et al., 2021), CD235a/GYPA (Abcam), HBD (Cell Signaling), HBG1/2 (Cell Signaling), and Flag (Sigma).

Techniques: Western Blot

Journal: eLife

Article Title: Modular UBE2H-CTLH E2-E3 complexes regulate erythroid maturation

doi: 10.7554/elife.77937

Figure Lengend Snippet: Figure 3. RANBP9 and RANBP10 form similar CTLH complex structures that cooperate with UBE2H to promote ubiquitin transfer. (A) Chromatograms (top) and Coomassie-stained SDS-PAGE gels (bottom) from size-exclusion chromatography of recombinant RANBP10-CTLHSR4 and RANBP9- CTLHSR4 complexes. (B) Cryo-EM map of RANBP10-CTLHSR4 (EMDB: EMD-16242, left) and RANBP9-CTLHSR4 (EMDB: EMD-12537) (right) with Cat- module and SRS-module indicated. (C) Focused refined map of the RANBP10-CTLH SRS-module with coloured subunits: ARMC8, purple; TWA1, salmon;GID4(Δ1–99), red; RANBP10 SPRY-domain, green. (D) Immunoprecipitation (IP) from HUDEP2 cell lysates with IgG control and UBE2H-specific antibody and immunoblot analysis. (E) IP from K562 cell lysates with IgG control and UBE2H-specific antibody and immunoblot analysis. IgG light chain (IgG-LC), IgG heavy chain (IgG-HC). (F) Fluorescence scan of SDS-PAGE gels presenting time course of in vitro ubiquitylation assay with fluorescently labelled model substrate peptide PGLW(X)n-23K with lysine at position 23 (pep*) in the presence of UBE2H, RANBP10-CTLH or RANBP9-CTLH, and GID4.

Article Snippet: For immunoblot analysis, lysates were denatured with SDS sample buffer, boiled at 95°C for 5 min, separated on SDS- PAGE, and proteins were visualized by immunoblotting using indicated primary antibodies: RMND5A (Santa Cruz), MAEA (R&D Systems), RANBP9 (Novus Biologicals), RANBP10 (Invitrogen, #PA5- 110267), TWA1 (Thermo Fisher), ARMC8 (Santa Cruz), WDR26 (Bethyl Laboratories), MKLN1 (Santa Cruz), YPEL5 (Thermo Fisher), GID4 (described in Sherpa et al., 2021), CD235a/GYPA (Abcam), HBD (Cell Signaling), HBG1/2 (Cell Signaling), and Flag (Sigma).

Techniques: Ubiquitin Proteomics, Staining, SDS Page, Size-exclusion Chromatography, Recombinant, Cryo-EM Sample Prep, Immunoprecipitation, Control, Western Blot, Fluorescence, In Vitro, Ubiquitin Assay