Rab11a antibody

The tissue sections were treated with xylene, graded alcohol. Antigen retrieval was performed in 0.01 M citrate buffer. H2O2 was used for blockage. Sections were treated with goat serum for 20 minutes. Then the slides was incubated Rab11a antibody (1:300 dilution, Proteintech, USA) overnight at 4°C. EliVision Super Kit (Maixin, Fuzhou, China) was then used for immunostaining. All tumor slides were examined randomly by two independent pathologists. Five views were selected in the center of tumor slides for evaluation. Rab11a staining was located in the cytoplasmic compartment of tumor cells. Immunostaining of Rab11a was scored following a semi-quantitative scale by evaluating the intensity and percentage of cells. The intensity of staining score was indicated as 0 (no staining), 1 (weak staining), 2 (strong staining). Staining percentage was scored as 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%) and 4 (76–100%). Each score was multiplied to a final score of 0 to 8. Rab11a status was regarded as low Rab11a expression (score < 4) or high expression/overexpression (score ≥ 4).

Tissue paraffin sections (5 μm) were deparaffinized using xylene and treated with graded alcohol (100%; 90%; 80%; 70%, 2 minutes each). Antigen retrieval was performed using citrate buffer (pH 6.0). Sections were then incubated with normal goat serum. Then sections were treated with Rab11a antibody (1 : 300 dilution, Proteintech, USA) overnight at 4°C. Immunohistochemical staining was performed using the Elivision plus kit (MaiXin, Fuzhou, China). Staining was developed with DAB plus kit (MaiXin, Fuzhou, China).
The slides were evaluated according to previous report [8 (link)]. Cytoplasmic localization was regarded as positive staining. Intensity was scored as 0 (no/weak staining), 1 (moderate staining), and 2 (strong staining). Score of staining percentage was classified as 1 : 1%–25%, 2 : 26%–50%, 3 : 51%–75%, and 4 : 76%–100%. Intensity and percentage scores were multiplied to the final score. Rab11a was considered low expression when the score was <4 and high expression when score was ≥4.

Total proteins from cell lines or fresh lung cancer/normal tissues were extracted in lysis buffer and quantified using the Bradford method. 30 mg protein was separated by SDS-PAGE. Nuclear/ cytoplasmic protein were separated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo scientific, USA) according to the manufacturer's protocol. Samples were transferred to PVDF membranes (Millipore, Billerica, MA, USA) and incubated overnight at 4°C with primary antibodies. Antibodies against cyclin D1, cyclin E, p27, CDK4, CDK6, CTGF, p-YAP, YAP (1:1000 dilution) and GAPDH (1:2000 dilution) were obtained from Cell Signaling Technology (Beverly, MA, USA). Rab11a antibody was from Proteintech (Proteintech, USA). After incubation with HRP-coupled anti-mouse or rabbit IgG antibody at 37°C for 2 hours. Target proteins on PVDF membrane were visualized using Pierce ECL kit and captured using a DNR BioImaging System (DNR, Jerusalem, Israel).
For immunoprecipitation, Magnetic Beads (Bio-Rad SureBeads) were incubated with antibodies and unbound antibodies were washed away. Then beads-antibody complex was incubated with target protein. The beads were magnetized using SureBeads magnetic rack and supernatant was discarded. Then elution buffer was used to collect purified target protein for western blot analysis.