rabbit polyclonal anti smn antibody  (Proteintech)

Journal: International Journal of Molecular Sciences

Article Title: The RNA-Binding Protein SMN as a Novel Player in Laryngeal Squamous Cell Carcinoma

doi: 10.3390/ijms24021794

Figure Lengend Snippet: SMN interacts with EGFR in laryngeal squamous cell carcinoma. ( A ) Cellular extracts from HLaC-79 cells were processed for co-immunoprecipitation assay (Co-IP) using EGFR polyclonal antibody-conjugated beads (IP-EGFR) or rabbit IgG-conjugated beads (IgG), as negative control. Then, samples were subjected to Western blot analysis. The 5% of the protein extract was used as input. Representative immunoblotting of three independent experiments, showing the co-precipitation of SMN with EGFR. ( B ) Representative image of in situ proximity ligation assay (PLA) performed in HLaC-79 cells using primary antibodies against SMN and EGFR (mouse monoclonal antibody and rabbit polyclonal antibody, respectively). PLA puncta (green dots) are indicative of SMN-EGFR interaction sites. Nuclei were labeled with DAPI (blue). Scale bar 10 μm. ( C ) Representative image of in situ proximity ligation assay (PLA) performed in de-paraffined sections of LSCC tissue from patient #5, using primary antibodies against SMN and EGFR (mouse monoclonal antibody and rabbit polyclonal antibody, respectively). PLA puncta (red dots) are indicative of SMN-EGFR interaction sites. As negative control, PLA was performed using only one of the primary antibodies (anti-SMN or anti-EGFR). Nuclei were labeled with DAPI (blue). Images were acquired with a 40× objective.

Article Snippet: The following antibodies were used: anti-SMN mouse monoclonal antibody (cat. no. 610647, BD Transduction Laboratories; work dilution for Western blotting, 1:10,000); anti-SMN rabbit polyclonal antibody (cat. no. sc-15320, Santa Cruz Biotechnology, Dallas, TX, USA; work dilution for immunofluorescence, 1:200); anti-SMN rabbit monoclonal antibody (cat. no. ab108424, Abcam, Cambrige, UK; work dilution for immunofluorescence, 1:200); anti-GAPDH mouse monoclonal antibody (cat. no. sc-47724, Santa Cruz Biotechnology; work dilution for western blotting, 1:500); EGFR rabbit polyclonal antibody (cat. no. sc-03-G, Santa Cruz Biotechnology; work dilution for immunofluorescence, 1:200); anti-Ki67 mouse monoclonal antibody (cat. no. MAB190, Millipore, Temecula, CA, USA; work dilution for immunofluorescence, 1:100); anti-RPS6 mouse monoclonal antibody (cat. no. sc-74459, Santa Cruz Biotechnology; work dilution for Western blotting, 1:1000; work dilution for immunofluorescence, 1:200); anti-E-cadherin mouse monoclonal antibody (cat. no. sc-71008, Santa Cruz Biotechnology; work dilution for Western blotting, 1:500); anti-vimentin mouse monoclonal antibody (cat. no. sc-6260, Santa Cruz Biotechnology; work dilution for immunofluorescence, 1:500); and anti-vimentin rabbit monoclonal antibody (cat. no. ab92547, Abcam; work dilution for immunofluorescence, 1:500).

Techniques: Co-Immunoprecipitation Assay, Negative Control, Western Blot, In Situ, Proximity Ligation Assay, Labeling

Journal: Biomolecules

Article Title: SMN Is Physiologically Downregulated at Wild-Type Motor Nerve Terminals but Aggregates Together with Neurofilaments in SMA Mouse Models

doi: 10.3390/biom12101524

Figure Lengend Snippet: SMN signal was high in motor synaptic terminals of control and SMA mice at P9-10. ( A ). Maximum intensity projections of TVA muscle motor terminals in both genotypes at P9. The motor endplates were identified using BTX-rhodamine labeling (red). Scale bars: 10 µm. ( B ). Comparison of the SMN area normalized to the postsynaptic area (BTX) in wild-type and SMNΔ7 mice (control and SMA). The analysis was performed at P9-10. ***: p < 0.01; n.s.: not significant; ( C , D ). Quantification and representative images of Western blot assays performed on TVA muscle extracts obtained from P10 wild-type (WT) and transgenic SMNΔ7 mice (control (Ctrl) and SMA). Either a mouse monoclonal antibody (mAb) anti-SMN or a rabbit polyclonal antibody (pAb) anti-SMN was used in the analysis. Note that with both the anti-SMN mAb ( C ) and the pAb ( D ), total-SMN levels were significantly increased in Ctrl, compared to WT (***: p < 0.001). In contrast, relative expression of total SMN in SMA mice was dramatically reduced compared with either WT or Ctrl animals (****: p < 0.0001 vs. WT or Ctrl (with the anti-SMN mAb); **: p < 0.01 vs. WT and ****: p < 0.0001 vs. Ctrl (with the anti-SMN pAb)). In all the cases, total-SMN levels (FL-SMN and SMNΔ7, when present) were quantified in 4 WT and in 8–11 Ctrl and SMA mice and normalized; first, to the loading control (GAPDH), and then, to the average of the WTs. Data are presented as mean ± SEM and were analyzed by using a one-way ANOVA (Bonferroni’s post hoc test). ( E , F ). Quantification and representative images of Western blot assays performed on TVA muscle extracts obtained from P10 and young adult (P35) WT and Ctrl transgenic SMNΔ7 mice, using either the anti-SMN mAb or pAb. Note that, with both the anti-SMN mAb ( E ) and the pAb ( F ), total-SMN protein levels at P35 were significantly lower than at P10, in WT and Ctrl mice (****: p < 0.0001 vs. P10 WT or Ctrl (with the anti-SMN mAb); **: p < 0.01 vs. P10 WT or Ctrl (with the anti-SMN pAb)). Moreover, at P35, SMN protein levels in TVA muscles remained significantly higher in Ctrl than in WT (*: p < 0.05 vs. WT (with the anti-SMN mAb); ****: p < 0.0001 vs. WT (with the anti-SMN pAb)). In all cases, total-SMN levels (FL-SMN and SMNΔ7, when present) were quantified in 4 mice per experimental condition and normalized; first, to the loading control (GAPDH), and then, to the average of the P10 WTs. Data are presented as mean ± SEM. Each two conditions were compared using a two-tailed Student’s t -test.

Article Snippet: Muscles were fixed in 4% PFA for 90 min, washed (0.1 M glycine in PBS) for 30 min, permeabilized (1% [ v / v ] Triton X-100 in PBS) for 90 min, and incubated in 5% ( w / v ) bovine serum albumin with 1% Triton X-100 in PBS for 3 h. Samples were incubated overnight at 4 °C, with the following primary antibodies: rabbit polyclonal anti-SMN H195 (sc-15320, SCBT), mouse monoclonal anti-NF-M (sc-51683, SCBT), and goat polyclonal anti-MAP1B (sc-8970, SCBT).

Techniques: Control, Labeling, Comparison, Western Blot, Transgenic Assay, Expressing, Muscles, Two Tailed Test