Home › Immunology › SorLA and CLC:CLF-1-dependent Downregulation of CNTFRα as Demonstrated by Western Blotting, Inhibition of Lysosomal Enzymes, and Immunocytochemistry
Immunology JoVE (Open Access) Citable · DOI

SorLA and CLC:CLF-1-dependent Downregulation of CNTFRα as Demonstrated by Western Blotting, Inhibition of Lysosomal Enzymes, and Immunocytochemistry

DOI: 10.3791/55019-v
What you'll learn
  • ✓ Perform Western blotting to detect CNTFRα protein downregulation
  • ✓ Apply immunocytochemistry with lysosomal inhibitors to track receptor trafficking
  • ✓ Interpret pSTAT3 levels as functional readout of receptor signaling
Protocol

The present protocol describes how Western blotting and immunocytochemistry combined with inhibition of lysosomal enzymes can be used to demonstrate the downregulation of Ciliary Neurotrophic Factor Receptor-α (CNTFRα), which is conveyed by the interaction between Cytokine-like Factor-1 (CLF-1) and the endocytic receptor sorLA.

Difficulty
advanced
Total time
~3–4 days (cell culture preparation, treatment, lysis, blotting, immunostaining, imaging)
Model organism
HEK293 cells (human embryonic kidney)
Biosafety
BSL-1

Steps

1
Prepare lysates and perform Western blotting

Extract protein from treated cells, separate by electrophoresis, and detect CNTFRα levels using antibodies to quantify receptor downregulation.

▶ 00:55
2
Perform immunocytochemistry with lysosomal inhibitors

Treat cells with lysosomal protease inhibitors, fix, stain for CNTFRα and relevant markers, and visualize subcellular localization to confirm lysosomal degradation pathway.

▶ 03:21
3
Detect pSTAT3 levels by Western blotting

Measure phosphorylated STAT3 protein as a downstream functional indicator of CNTFRα signaling activity in response to CLF-1/CLC treatment.

▶ 06:00
4
Analyze and interpret downregulation results

Combine Western blot densitometry, immunofluorescence quantification, and pSTAT3 data to confirm sorLA and CLF-1-mediated CNTFRα downregulation.

▶ 08:17
💬 Comments coming soon