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The Norepinephrine Transporter (NET) is encoded by the *SLC6A2* gene and belongs to the family of sodium-dependent neurotransmitter symporters; it is a multi-spanning transmembrane protein featuring 12 to 13 transmembrane domains. NET is primarily expressed in the brainstem, adrenal glands, and the presynaptic membranes of sympathetic neurons. Its core physiological function is to efficiently reuptake norepinephrine from the synaptic cleft back into the presynaptic nerve terminal, thereby terminating its signal transmission. Clinically, loss-of-function mutations in the *SLC6A2* gene can lead to orthostatic intolerance. Furthermore, this gene serves as a primary therapeutic target for various psychoactive drugs, such as the tricyclic antidepressant desipramine, as well as stimulants like cocaine and amphetamine.

Fundamentally, NET acts as a transporter rather than a classical signaling receptor; its core function is to clear norepinephrine from the synaptic cleft via a mechanism of secondary active transport. This process is strictly dependent on the concentration gradients of Na⁺ and Cl⁻; by co-transporting Na⁺, Cl⁻, and the substrate into the cytoplasm, it terminates norepinephrine signaling and facilitates the recycling of monoamines. NET activity is subject to precise regulation: at the transcriptional level, neuronal depolarization can induce the upregulation of *SLC6A2* gene expression—a process involving the dissociation of epigenetic regulators such as MeCP2 and Smarca2, as well as the recruitment of SP1. At the post-translational level, various protein kinases (e.g., PKC, p38 MAPK) can influence the surface expression levels of NET through phosphorylation, thereby rapidly modulating its transport capacity. Additionally, numerous drugs exert their pharmacological effects by directly binding to NET; for instance, the antidepressant desipramine blocks substrate transport by binding to the inner end of the transporter's extracellular vestibule, thereby locking the extracellular gate.

The NET (*SLC6A2*) CHO cell-based drug target model effectively simulates the *in vivo* function of the transporter in regulating neuronal signal transduction; the underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the NET (*SLC6A2*) CHO Cell Model

Figure 2. WB of NET(SLC6A2) CHO.

Figure 3. Inhibition of NET inhibitor in NET(SLC6A2) CHO(C15).

Cell Resuscitation
1)Rapidly thaw the frozen cells in a 37 °C water bath for approximately 60 seconds. Once thawed (which may take slightly less or more than 60 seconds), immediately transfer the cell suspension from the cryovial into a 15 mL centrifuge tube containing 10 mL of pre-warmed CHO-K1 Human NET(SLC6A2)  Cell complete culture medium.
2)Centrifuge cells at 1000 rpm for 5 min to remove medium, then resuspend cells in 5 mL of pre-warmed complete medium.
3)Transfer the cell suspension into a T25 culture flask and incubate at 37 °C with 5% CO₂.
4)After approximately 24–36 hours, replace the medium or passage the cells to remove non-adherent dead cells.

Subculturing procedure
1)When the cell density reaches the appropriate confluency for passaging, wash the cells with PBS, then add 1 mL trypsin to detach the cells. When more than 80% of the cells detach upon gently tapping the culture flask, add complete culture medium to terminate digestion. Gently pipette to obtain a single-cell suspension, transfer to a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 5 minutes.

2)Discard supernatant after centrifugation. Resuspend cells in fresh medium to a single-cell suspension and transfer to a new culture flask for continued growth.

Cell Freezing
After trypsinization and centrifugation of cells from each T75 flask or 10 cm culture dish, discard the supernatant. Add 2 mL of cryopreservation medium (90% FBS + 10% DMSO), gently resuspend thoroughly, and aliquot into two cryovials. Immediately place the cryovials into a controlled-rate freezing container (e.g., Nalgene 5100-0001), fill with isopropanol to the indicated level, and store at −80 °C. After 24 hours, transfer the cryovials to liquid nitrogen for long-term storage.