Sc 48345

Non-commercial, affinity-purified rabbit-raised polyclonal antibody against the C-terminal peptide of mSglt1 protein (mSglt1-Ab) was previously described [21 (link)]. Commercial polyclonal anti-SGLT1 antibodies were from Merck Milipore (07-1417, Lot no. 2730935), Abcam (ab14686), and Santa Cruz Biotechnology (M-19, sc-20582). According to information in the respective technical sheets, these polyclonal antibodies are supposed to react with the human and rodent SGLT1/Sglt1 proteins. Monoclonal antibodies against actin (actin-Ab) and Na⁺/K⁺-ATPase (Na/K-ATPase-Ab) were purchased commercially from Merck Milipore (MAB1501R) and Santa Cruz Biotechnology (sc-48345), respectively; their use was described in our recent publication [54 (link)]. Secondary antibodies included the alkaline phosphatase-labeled goat anti-mouse IgG (GAM-AP) and goat anti-rabbit IgG (GAR-AP) (both from Jackson ImmunoResearch Laboratories, USA) for Western blotting, as well as the CY3-labeled goat anti-rabbit IgG (GAR-CY3) and donkey anti-goat IgG (DAG-CY3) IgG (Jackson ImmunoResearch Laboratories, USA), and FITC-labeled goat anti-mouse IgG (GAM-FITC; Kirkegaard & Perry, USA) for immunocytochemistry.

The HaCaT cells were seeded in six-well confocal plates at a density of 1 × 10⁶/well. After 24 h of incubation, the cells were washed twice with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 5 min at 37 °C. Then, the cells were incubated with 5% bovine serum albumin dissolved in PBS for 45 min at 37 °C. The HaCaT cells were incubated overnight with one of the following primary antibodies for immunocytochemistry: OR2AT4 (PA5-39811, 1:200; Thermo Fisher Scientific), sodium/potassium-ATPase α (Na⁺/K⁺-ATPase-α) (SC-48345, 1:50; Santa Cruz Biotechnology), and Ki-67 (NB500-170, 1:200; Novus). Then, the cells were washed three times with PBS. The HaCaT cells were incubated with anti-mouse IgG (Alexa Fluor 488; A11001, 1:500; Thermo Fisher Scientific) or anti-rabbit IgG (Alexa Fluor 532; A11009, 1:500; Thermo Fisher Scientific) secondary antibody for 1 h at room temperature. Next, the cells were incubated with 300 nM DAPI (Cayman Chemical, Ann Arbor, MI, USA) for 5 min in the dark. The cells were washed twice with PBS. Images were obtained using the LSM510 META confocal microscope and analyzed using LSM700 software (version 3.2; Carl Zeiss, Jena, Germany).

The labeling of surface GABA_A receptors and confocal immunofluorescence microscopy analysis were performed according to published procedure [14 (link)]. Briefly, cells on coverslips were fixed for 3 min with 4% formaldehyde on ice and incubated in 100 μL of HEPES buffer (HEPES 25 mM, NaCl 140 mM, KCl 5.4 mM, CaCl₂ 1.8 mM, glucose 15 mM, pH = 7.4) containing mouse monoclonal anti-α1 antibody (Millipore #MAB339) and rabbit monoclonal anti-Na⁺/K⁺-ATPase, a plasma membrane marker (Abcam #ab76020), or rabbit polyclonal anti-γ2 antibody (Synaptic Systems #224,003) and mouse monoclonal anti-Na⁺/K⁺-ATPase, a plasma membrane marker (Santa Cruz Biotechnology #sc-48345) for 1.5 h. The primary antibodies were used at 1:300 dilutions. Cells were then incubated with 100 μL of HEPES buffer containing an Alexa 488-conjugated secondary antibody (1:600) and an Alexa 594-conjugated secondary antibody (1:600). Afterwards, cells were permeabilized with saponin (0.2%) for 10 min and incubated with DAPI (1 µg/mL) for 3 min to stain the nucleus. The coverslips were then mounted and sealed. For confocal immunofluorescence microscopy, an Olympus IX-81 Fluoview FV1000 confocal laser scanning system was used. A 60X objective collected images using FV10-ASW software. Quantification of the fluorescence intensity after background correction was done using the ImageJ software from the NIH.