Iblot transfer stack

After 48 hr of cell culture, CD4⁺ T cells were harvested and lysed using RIPA buffer with protease and phosphatase inhibitors. BCA Protein Assay Kit (Thermo Fisher Scientific, Cat# 23225) was used to determine protein concentration according to the manufacturer’s instructions and samples adjusted to contain 30 μg of protein in Laemmeli Sample Buffer (Sigma-Aldrich, Cat# S3401) containing β mercaptoethanol (Sigma-Aldrich, Cat# 63689). Cell lysates were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose mem- branes using iBlot Transfer Stack (Invitrogen, Cat# IB301002) and Thermo Fisher Scientific iBlot Gel Transfer Device. The membranes were then incubated with primary anti phospho-p70S6K, followed by secondary antibody goat anti-Rabbit. Bands were detected using Clarity Western ECL Substrate (Bio Rad, Cat# 1705061) and imaged using the Bio Rad ChemiDoc Imaging System.

CD8 T cells were centrifuged at 1000 xg for 5 minutes and supernatant was removed. Cells were then washed with PBS twice before resuspending them in an appropriate volume of RIPA lysis buffer (50mM TRIS pH 8.0, 150mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with HALT Protease and Phosphatase inhibitor cocktail (78444, Fisher Scientific). Lysis was carried out on ice for 30 minutes. Samples were then centrifuged at 17000 xg and whole cell lysates were collected. Bio-Rad protein assay (500-0006, Bio-Rad) was used to quantify the protein content of each lysate. Protein concentrations were equalized and immunoblot samples were prepared by addition of 4xLDS sample buffer (15484379, Fisher Scientific) containing 10% b-Mercaptoethanol (final concentration 2.5%) and subsequent incubation of the samples at 95°C for five minutes. Proteins in lysates were size-separated on 4-12% Bis-Tris polyacrylamide-SDS gels (Invitrogen) and transferred to iBlot™ Transfer Stack (Invitrogen). Blots were blocked using 4% BSA in 0.2% Tween-20 in PBS. Blocked membranes were incubated with primary antibodies overnight. Immunoblots were developed using the Super Signal West Dura Extended Duration Substrate (34075, Thermo Fisher Scientific). The luminescence signal was captured by the Bio-Rad ChemiDoc imaging system. See key resources table for antibody list.

The MNC (2 × 10⁷) were resuspended in cell lysis buffer (NP40 Cell Lysis Buffer, Invitrogen) and subjected to SDS-PAGE (Invitrogen). The protein bands were transferred to a nitrocellulose membrane using iBlot® Transfer Stack (Invitrogen) and subjected to immunoblot analysis with the antibodies. Anti-p38 (no. 8690), p-p38 (no. 4511), ERK (no. 4695), p-ERK (no. 4370), p-NF-kB p65 (no. 3033), IkBα (no. 4814), β-actin (no. 4970) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). The alkaline phosphatase (AP) linked anti-rabbit IgG (no. 550321B), anti-mouse IgG (no. 550321A), AP chemiluminescent substrate (no. 1208012) and its enhancer were purchased from Invitrogen.

Cells were homogenized and protein concentrations were measured by the Bradford method. Lysed proteins of each sample were separated by a Bolt™ 4–12% Bis-Tri Plus gel and transferred onto nitrocellulose membranes using an iBlot Transfer Stack (Invitrogen, Carlsbad, CA, USA). After blocking with 5% non-reduced fat milk in TBST for 1 h, the membranes were incubated overnight at 4 °C with the following primary antibodies: PP1CB (1:500), cyclin D1 (1:10,000), phospho-cyclin D1 (Thr 286, 1:1000), pRB, phospho-pRB (Ser780, 1:1000) (Cell Signaling Technology, Danvers, MA, USA) and p16 (1:5000), p21 (1:5000) (Abcam, Cambridge, MA, USA). The secondary antibody, F (ab’) 2-goat anti-rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody (1:10,000) (Invitrogen, Carlsbad, CA, USA), was used at room temperature for 1 h incubation. The blot was visualized by using Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, Rockford, IL, USA). Each band was quantified via ImageJ software, and the value was normalized to loading control by Ponceau (Sigma-Aldrich, Darmstadt, Germany).