Anti flag tag antibody

Huh7 (1 × 10⁷/200 μL) cells were injected subcutaneously into the right axilla of BALB/c nude mice (Vital River Laboratories, Beijing, China). When the tumors grew to average diameter of 5 mm, the animals were randomized into 5 groups and received intravenous injection of either PBS, CIK cells, KGHV500, CIK + KGHV500, or CIK + KGHV400, respectively. Tumor size was measured with Vernier caliper every three days. Tumor volume was calculated according to the formula: V= (length × width²) ÷ 2 16 (link). When tumors in PBS group became ulcerated, mice were sacrificed. The dissected tumors were embedded with paraffin for immunohistochemical analysis. The anti-FLAG tag antibody (Abnova, MAB9744, China) and anti-human adenovirus hexon mouse mAb (Novus, NBP211638, USA) were used to detect KGHV500 adenovirus.

Prokaryotically expressed K-p21Ras [43 (link)] was separated by SDS-PAGE, then transferred to polyvinylidene fluoride (PVDF) membranes and incubated with RGD4C-p21Ras-scFv. Next, the PVDF membranes were incubated with anti-Flag tag antibody (Abnova, #2368, China). Subsequently, the membranes were washed and incubated with a goat anti-mouse/rabbit IgG antibody and horseradish peroxidase (HRP) (ZSGB-Bio, ZB-5305, China) at 37 °C for 45 min. After washing with TBST, the protein bands were visualized with a 3,3′-diaminobenzidine (ZSGB-Bio) [29 ].

WB assay was performed by extracting protein from cell samples with RIPA cellular lysate (Solarbio), followed by electrophoresis on SDS-PAGE gels and transfer of proteins to polyvinylidene fluoride (PVDF) membranes. Next, the PVDF membranes were incubated with BR2-p21Ras scFv (0.3mg/ml,dilution1:800) after using skimmed milk powder as a blocking agent and then were incubated with the anti-FLAG tag antibody(Abnova,MAB9744,China) at a 1:1000 dilution and horseradish peroxidase (HRP) labeled goat anti-mouse IgG (ZSGB-Bio, ZB-5305, China) at 1:1000. After washing with TBST (50mMTris, 150mMNaCl, and 0.5%Tween-20)for three times, The PVDF membranes were then stained with DAB. β-Actin (ZSGB-Bio,TA-09,China) protein was used as an internal control.