I have been treated PC12 cells with 100ng/ml NGF for 10 days, and lysed them using RIPA buffer for western blot. I got no bands for primed cells, but for wild type cells. Does anyone know why?!
Hi Rojin Falah
The absence of TrkA overexpression in NGF-treated PC12 cells doesn't contradict the observed neurite outgrowth due to: Receptor Downregulation: Chronic NGF exposure can decrease TrkA levels due to feedback inhibition. Timing of Protein Expression: TrkA overexpression may occur at an earlier time point and decrease by 10 days. Detection Sensitivity: Ensure optimal Western blotting protocol for efficient detection. Post-translational Modifications: Some antibodies might not detect phosphorylated or other modified forms of TrkA. Experimental Condition Differences: Ensure similar cell lysis conditions and protein normalization.
The functional outcome of NGF treatment is associated with TrkA activation and downstream signaling, not necessarily with increased TrkA expression.
Good luck!
The lack of TrkA overexpression in pretreated PC12 cells compared to wild-type (WT) PC12 cells, despite observing neurite outgrowth in response to NGF, could be due to several reasons:
1. Saturation of TrkA Expression: In untreated WT PC12 cells, TrkA receptors might already be expressed at their maximum level. Additional NGF treatment may not further induce TrkA overexpression, resulting in no observable change in TrkA levels in the Western blot.
2. Downstream Signaling: NGF can activate signaling pathways beyond TrkA, leading to neurite outgrowth through alternative mechanisms. Neurite outgrowth can occur via other receptor systems or downstream signaling pathways, which may not involve TrkA upregulation.
3. Time Point of Analysis: The time point you selected for Western blot analysis might not be optimal for capturing changes in TrkA expression. TrkA levels may fluctuate transiently in response to NGF, and 10 days of treatment may not coincide with a peak in TrkA expression.
4. Differentiation Stage: PC12 cells have the ability to differentiate into neuronal-like cells. The mechanism of neurite outgrowth might be related to differentiation rather than TrkA overexpression specifically.
5. Post-Transcriptional Regulation: TrkA expression could be regulated at the post-transcriptional or post-translational level. Even if TrkA mRNA levels remain unchanged, protein expression may be modulated by various regulatory processes.
6. Technical Issues: There could be technical problems during the Western blot procedure, such as inadequate sample loading, insufficient sensitivity of the antibody, or issues with protein extraction using RIPA buffer, which might affect the detection of TrkA.
To gain a better understanding of the underlying mechanism of neurite outgrowth in your pretreated PC12 cells, you may consider the following:
- Verify the differentiation status of the PC12 cells by performing additional markers analysis specific to neuronal differentiation.
- Try different time points for Western blot analysis to capture potential changes in TrkA expression during the NGF treatment.
- Employ alternative techniques like immunofluorescence or flow cytometry to assess TrkA expression at the cellular level.
- Explore other downstream signaling molecules or markers associated with neurite outgrowth in the pretreated PC12 cells.
All the best
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