Pkcα antibody

DR peptide (DVEDSYGQQWTYEQR) was purchased from 1st Base, Singapore, which was used to raise the DR-Ab and also to block DR-Ab. PKCα antibody and phosphor-GluR2ser880 antibody were purchased from Cell Signalling (Danvers, MA 01923, USA). GluR2 antibody was purchased from Abcam. NKAα antibody (H-3, sc-48345), NKAα1 antibody (sc-21712), NKAα2 antibody (sc-31391), NKAα3 antibody (sc-58631), EAAT1 antibody (Cell Signaling Technology, #5684), EAAT2 antibody (Cell Signaling Technology, #3838), goat anti-rabbit, and goat anti-mouse secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA95060, USA). Fura-2 AM, Fluo-4 AM, Alexa Fluor 568 conjugated goat anti-rabbit IgG (H+L), and Alexa Fluor 488 conjugated goat anti-mouse IgG (H+L) were from Invitrogen Corporation (Carlsbad, CA, USA).

Flow cytometry analysis of cell-surface receptor CCR6 detection (using anti-CCR6, cat# 559562, BD Bioscience) was performed at 72 h as described earlier (15 (link)). To measure PKCα overexpression at protein level, FACS samples were collected six hours after in vitro transcribed PKCa RNA had been introduced to cells by nucleofection. The cells were fixed using pre-warmed BD Phosflow Fix buffer I (BD, Cat. no. 557870) for 10 minutes at 37°C. The cells were collected after centrifugation and resuspended in 300ul of cold Perm buffer III and incubated at +4°C for 30 minutes. Perm buffer was removed and cells were washed in 200ul of Stain buffer (BD Cat#554656). The PKCα antibody (Cell signaling Cat# 59754) was used in 1:100 dilution in staining buffer and the samples were incubated at room temperature for 30 minutes. Cells were washed two times and resuspend in staining buffer containing Alexa fluor-647 secondary antibody (cell signaling Cat no. 4414) for 30 minutes at room temperature. Cells were washed for two times and resuspend in PBS for FACS analysis. The data was acquired in BD LSR II/LSRFortessa. The acquired data were then analyzed either by Flowjo (FlowJo LLC) or Flowing software (https://bioscience.fi/services/cell-imaging/flowing-software/).

Proteins were extracted from liver tissues and HepG2 cells using a cell membrane protein and plasma protein extraction kit (Beyotime, P0033), and the following antibodies were used for Western blot (WB) analyses: BSEP polyclonal antibody (PA5‐78690, Invitrogen, Carlsbad, CA, USA); caveolin‐1 antibody (Cell Signaling Technology, Inc., Danvers, MA, USA); anti‐Hax‐1 antibody (Ab137613, Abcam plc., Cambridge, UK); PKCα antibody (Cell Signaling Technology, Inc.); phospho‐PKCα/β II (Thr638/641) antibody (Cell Signaling Technology, Inc.); mouse anti‐GAPDH antibody (Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China); Na,K‐ATPase antibody (Cell Signaling Technology, Inc.); and mouse anti‐β actin antibody (Zhongshan Golden Bridge Biotechnology Co., Ltd.). Protein expression was normalized to β‐actin, GAPDH and Na⁺, K⁺‐ATPase. Densitometry analyses were performed using the ImageJ software.
¹⁴ (link)