Journal: EMBO Reports

Article Title: Muskelin is a substrate adaptor of the highly regulated Drosophila embryonic CTLH E3 ligase

doi: 10.1038/s44319-025-00397-6

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rabbit anti-eIF4E (Boster) was used at 1:10000.

Techniques: SYBR Green Assay, Imaging, Recombinant, Sequencing, Software, Microscopy, Ion-Mobility Spectrometry, Mass Spectrometry

Journal: Cancer gene therapy

Article Title: Targeting and killing of prostate cancer cells using lentiviral constructs containing a sequence recognized by translation factor eIF4E and a prostate-specific promoter.

doi: 10.1038/sj.cgt.7700885

Figure Lengend Snippet: Figure 3 Western analysis of eIF4E level in cancer and noncancer cell lines. Protein extracts from cancer cells (LNCaP, MCF-7, PC- 3M, and DU145) and noncancer cells (BPH-1, Plat-E, Huvec-c, and 267-B1) were probed with antibodies to eIF4E and b-actin in a Western blot. Density scans (means7s.e.m., n ¼ 3) of eIF4E/b-actin are shown in the bottom panel.

Article Snippet: The membranes were blocked with 5% skim milk in trisbuffered saline (TBS) containing 0.05% tween-20 and probed with 1:1000 rabbit polyclonal anti-eIF4E antibody (Cell Signalling), 1:500 anti-HTK (a gift from Dr William C Summers, Yale University), or 1:500 anti-b-actin antibody (Sigma), followed by 1:2000 polyclonal goat anti-rabbit antibody conjugated to horseradish peroxidase (Bio-Rad).

Techniques: Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Cytoplasmic poly(A)-binding protein 1 (PABPC1) interacts with the RNA-binding protein hnRNPLL and thereby regulates immunoglobulin secretion in plasma cells

doi: 10.1074/jbc.M117.794834

Figure Lengend Snippet: hnRNPLL interacts with PABPC1 A, mass spectrometric analysis identifies interaction between hnRNPLL and PABPC1. hnRNPLL was immunoprecipitated (IP) from MPC11 cells, and precipitated proteins were resolved on SDS-PAGE and stained with Coomassie Blue. The indicated protein bands were then exercised and analyzed by mass spectrometry. The identified proteins are as follows: top band, the hnRNPLL long isoform, which utilizes an alternative translational start site (17); middle band, PABPC1; lower band, the canonical hnRNPLL protein. B and C, PABPC1 interacts with hnRNPLL in lymphocytes. As in A, hnRNPLL (LL) was immunoprecipitated from MPC11 (B) or Jurkat cells (C), and the immunoprecipitate was analyzed for the presence of hnRNPLL, PABPC1, eIF4G2, and eIF4E by immunoblotting (IB) as indicated. Normal rabbit IgG (IgG) was used as a negative control for immunoprecipitation, and 5% of the total protein lysate was included as input control. D, hnRNPLL co-immunoprecipitates with PABPC1. FLAG-tagged PABPC1 was transduced into MPC11 cells, FLAG-PABPC1 was immunoprecipitated with an anti-FLAG antibody (M2), and the immunoprecipitate was analyzed for FLAG, hnRNPLL, and eIF4G2 by immunoblotting as indicated. Normal mouse IgG was used as negative control, and 3% of the total protein lysate was included as input control. E, hnRNPLL interacts with PABPC1 both in the nucleus and in the cytoplasm. MPC11 cells were co-stained with anti-PABPC1 (red) and anti-hnRNPLL (green) and analyzed by confocal immunofluorescence microscopy. Note that hnRNPLL was predominately observed in the nucleus with notable cytoplasmic localization; in contrast, PABPC1 was mainly in the cytoplasmic and was also presented in the nucleus. F, PABPC1 is located in both the nucleus and the cytoplasm of MPC11 cells. MPC11 cells were fractionated into cytoplasmic (C) and nuclear (N) fractions, and the expression of PABPC1 and hnRNPLL was analyzed by immunoblotting. GAPDH and lamin B were served as the controls for cytoplasmic and nucleus proteins, respectively. C, cytoplasmic fraction; N, nucleus fraction. G, both isoforms of hnRNPLL can interact with PABPC1. The FLAG-tagged hnRNPLL constructs were transfected into HEK 293 T cells, and hnRNPLL was immunoprecipitated from the cell lysate with the anti-FLAG (M2) antibody. The immunoprecipitate was analyzed for PABPC1 and hnRNPLL. 1% of the total protein lysate was included as input control. H, PABPC1 interacts with both isoforms of hnRNPLL. PABPC1 fused with a Myc tag was transfected into HEK 293 T cells alone or with either of the hnRNPLL isoforms. PABPC1 was immunoprecipitated from the cell lysate with an anti-Myc antibody, and the immunoprecipitate was analyzed for hnRNPLL and Myc. 1% of the total protein lysate was included as input control. B–H, data are representative of at least three independent experiments.

Article Snippet: Proteins were separated on 8 or 10% SDS-PAGE gel, and immunoblotting was performed with anti-mouse hnRNPLL, anti-human hnRNPLL (ab74063; Abcam), anti-PABPC1 (orb34329; biorbyt), anti-eIF4G2 (AB60254a; BBI), anti-ELL2 (12727-1-AP; Proteintech), anti-eIF4E (AB55221; BBI), anti-lamin B (sc-365962; Santa Cruz), anti-FLAG (F7425; Sigma), anti-Myc (AT0023; CMCTAG), and anti-GAPDH (sc-48166; Santa Cruz).

Techniques: Immunoprecipitation, SDS Page, Staining, Mass Spectrometry, Western Blot, Negative Control, Immunofluorescence, Microscopy, Expressing, Construct, Transfection

Journal: bioRxiv

Article Title: Precise temporal regulation of post-transcriptional repressors is required for an orderly Drosophila maternal-to-zygotic transition

doi: 10.1101/862490

Figure Lengend Snippet: A-B. FLAG IP-MS of 0-3h embryo lysate collected from transgenic flies expressing FLAG-SMG, and homozygous for the deletion allele smg 47 . FLAG IP from non-transgenic embryo lysate was used as control. Average spectral counts are plotted for proteins detected at ≥1 in FLAG-SMG IP on average across at least 4 biological replicates. A. In the absence of RNase A, SMG interacts strongly with its co-repressors (red). B. IP in the presence of RNase A captured RNA-independent interactions with two E3 ubiquitin ligase complexes: the CTLH complex (blue: Muskelin, RanBPM, CG6617, CG3295, CG31357 and CG7611), and the SCF complex (green: CUL1, SKPA, CG14317 and SLMB). C-D. GFP IP-MS of 0-2h embryo lysate from embryos expressing either GFP-Muskelin or GFP-SLMB. GFP IP from non-transgenic embryo lysate was used as control. Average iBAQ intensities for proteins detected across 3 biological replicates are plotted. C. GFP-Muskelin interacts with the RBPs and other members of the CTLH complex. D. GFP-SLMB interacts with the RBPs and other members of the SCF complex. Note: Cutoffs were not applied to negative controls. Proteins not detected in control IPs are assigned an average spectral count of 0.1 (A-B), or an iBAQ value of 10 (C-D) to avoid log(0); these small values are at least twofold less than the lowest detected values across all experiments.

Article Snippet: Anti-eIF4E and anti-CG3295 antibodies were raised in rat (Eurogentec).

Techniques: Transgenic Assay, Expressing

Journal: bioRxiv

Article Title: Precise temporal regulation of post-transcriptional repressors is required for an orderly Drosophila maternal-to-zygotic transition

doi: 10.1101/862490

Figure Lengend Snippet: RT-qPCR quantification of target mRNA expression in 0-3h embryos. A. muskelin , B. ranBPM and C. CG3295 mRNAs were depleted by > 90% relative to mCherry control RNAi in their respective maternal knockdowns.

Article Snippet: Anti-eIF4E and anti-CG3295 antibodies were raised in rat (Eurogentec).

Techniques: Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Precise temporal regulation of post-transcriptional repressors is required for an orderly Drosophila maternal-to-zygotic transition

doi: 10.1101/862490

Figure Lengend Snippet: Quantified developmental western blots of RBP expression. Embryos were collected from maternal knockdown of CTLH complex members over the first four hours after egg-lay. Knockdown of muskelin, ranBPM and CG3295 each independently resulted in significant stabilization of Cup ( A ), TRAL ( B ) and ME31B ( C ) relative to control mCherry knockdown. D. SMG protein degradation was unaffected by knockdown of the CTLH complex. * P < 0.05, n.s. = not significant, n = 3, error bars = SD, S tudent’s t -test. Knockdown was confirmed by qPCR , see also - .

Article Snippet: Anti-eIF4E and anti-CG3295 antibodies were raised in rat (Eurogentec).

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: Precise temporal regulation of post-transcriptional repressors is required for an orderly Drosophila maternal-to-zygotic transition

doi: 10.1101/862490

Figure Lengend Snippet: A-D. Western blots of embryos picked at Stage 2, Stage 5 and Stage 7 (n = 3 biological replicates for each stage). Maternal knockdown of muskelin ( A ), ranBPM ( B ) and CG3295 ( C ) resulted in stabilization of Cup, TRAL and ME31B through embryo Stage 7, whereas all three RBPs were depleted in the embryo by Stage 5 in the control knockdown ( D ) . E-G. RT-qPCR quantification of mRNA expression in embryos assayed in A-D. Picking developmentally stage-matched embryos partially rescued the delay in mRNA degradation in CTLH maternal knockdown embryos. cup ( E ) was cleared to comparable levels as in control mCherry RNAi by stage 7. tral ( F ) and me31B ( G ) remained partialized stabilized. * P < 0.05, n.s. = not significant, n = 3, error bars = SD, Student’s t -test.

Article Snippet: Anti-eIF4E and anti-CG3295 antibodies were raised in rat (Eurogentec).

Techniques: Western Blot, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Precise temporal regulation of post-transcriptional repressors is required for an orderly Drosophila maternal-to-zygotic transition

doi: 10.1101/862490

Figure Lengend Snippet: A. Expression of subunits of the CTLH complex captured in the developmental proteome. Most subunits have relatively constant levels throughout embryogenesis, while levels of Muskelin are highest at the first time point and then decrease rapidly. B. Western blot of embryos expressing GFP-Muskelin. Anti-GFP (top) confirmed rapid clearance of GFP-Muskelin from the embryo. Anti-CG3295 (bottom) confirmed its stable expression during the MZT. C. Expression of subunits of the SCF complex captured in the developmental proteome. Most subunits exhibit relatively constant levels throughout embryogenesis, while levels of the F-box subunit, CG14317 increased rapidly during the MZT and then decreased very rapidly by the end of the MZT, with peak levels coinciding with degradation of SMG protein. D. Developmental western blot of control RNAi embryos, confirming the stable expression of SLMB during the MZT. Note: The same blot shown here is used to confirm SLMB knockdown in .

Article Snippet: Anti-eIF4E and anti-CG3295 antibodies were raised in rat (Eurogentec).

Techniques: Expressing, Western Blot