Lc3b antibody

HepG2.2.15 cells containing transfected HBV full-length DNA [17 (link)] or HepG2 cells were treated with 200 nmol/L Bortizomib (Millennium pharmaceuticals, USA) alone or in combination with 100 nmol/L Rapamycin (Enzo Life Sciences, China) or 30 mmol/L NH₄Cl for 18 h, DRibbles containing autophagosomes were prepared from the culture media as described [13 (link),14 (link)]. Morphology analysis of HBV⁺ DRibbles was done under transmission electron microscopy. HBsAg in HepG2.2.15 cell lysates was detected by western blot analysis using anti-HBsAg antibody (Santa Cruz, Biotechnology, Inc., USA). LC3 in both HepG2.2.15 cell lysates and HBV⁺ DRibbles was determined by western blot analysis using the polyclonal LC3B antibody (Cell Signal Technology, USA,1:1000), the HRP-labeled goat anti-rabbit IgG secondary antibody (1:5000) and a chemiluminescence kit (Multisciences Biotech Co., Ltd., China). Levels of HBsAg and HBeAg in HBV⁺ DRibbles were measured by ELISA (Shanghai Kehua Bioengineering Co., Ltd., China).

Arginase-1 (ARG-1) rabbit antibody (1:700) was from Abcam (Cambridge, UK); CD163 rabbit antibody (1:100) was from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); LC3B antibody (1:500); mTOR (1:500) and p-mTOR (1:500) rabbit antibodies were from Cell Signaling Technology Inc. (Beverly, MA, USA). SQSTM1/p62 mouse monoclonal antibody (1:500) was obtained from Abcam (Cambridge, UK). GAPDH mouse antibody (1:2500) from Calbiochem (San Diego, CA, USA) was used for protein normalization. HRP-linked anti-rabbit and anti-mouse antibodies were from Jackson Immunoresearch (West Grove, PA, USA).

The gastrocnemius muscle homogenate was centrifuged at 10,000g for 10 min at 4 °C. The supernatant was mixed with an equal volume of 2 × SDS sample buffer, and the diluted gastrocnemius muscle sample was subjected to electrophoresis on a 7.5 % polyacrylamide gel for S6K1 analysis or a 4–15 % gradient gel (Bio-Rad, Hercules, CA) for LC3B and GAPDH analyses. The samples were subjected to protein immunoblot analysis using S6K1 polyclonal antibodies (dilution 1:500, sc-230, Santa Cruz Biotechnology, Santa Cruz, CA), LC3B antibody (#2775, Cell Signaling Technology, Danvers, MA) or GAPDH antibody (#2775, Cell Signaling Technology). The S6K1 phosphorylation ratio was quantified using the ratio of the heavier phosphorylated forms (β + γ forms) to total immune reactivity (α + β + γ forms), because S6K1 resolves into multiple electrophoretic forms as a result of reduced electrophoretic mobility with increased phosphorylation, as described previously (Kimball et al. 1998 (link), Yoshizawa et al. 2001 (link)). The protein content was normalized to GAPDH in the LC3B analysis.

After treatment, LAPC4-KD cells were washed and fixed with 1% paraformaldehyde (Sigma-Aldrich) followed by blocking with 1% BSA for 1 h. Cells were probed with LC3B antibody (Cell Signaling) at room temperature for 1 h and then incubated with Alexa Fluor 555-conjugated anti-rabbit antibody (Invitrogen) for 1 h (Xu et al., 2007 (link)). Nuclei were counterstained with Hoechst 33342 for 10 min. The stained cells were then analyzed using a fluorescence microscope (Carl Zeiss) as described previously (Liao et al., 2017 (link)).

Tumor cells were seeded in a T175 flask in complete RPMI-1640 culture medium supplemented with 10% heat-inactivated FBS (Gibco), 100 U/ml penicillin, and 0.1 mg/ml streptomycin and incubated for 3–4 days at 37 °C, 5% CO₂ until 100% confluency was reached. Tumor cell culture supernatants were collected for TRAPs isolation as described previously [18 (link), 20 (link)]. Briefly, supernatants were centrifuged at 2000 rpm for 10 min to remove whole cells and debris. The supernatants were further centrifuged at 12,000 g for 30 min to harvest the TRAPs-containing pellet. The TRAPs-containing pellet was washed three times with PBS and isolated with magnetic beads (Miltenyi Biotec) combined with LC3b antibody (Cell Signaling Technology) for TRAPs. The purity of TRAPs was analyzed by flow cytometry and western blot. The size of TRAPs was determined by dynamic light scattering using a Malvern Instrument.

Approximately 5,000 PTX-treated cells were seeded in 24-well plates and fixed. After permeabilization with Triton-X-100 for 15 min, the cells were blocked by 5% goat serum for 1 h and incubated with the LC3B antibody (1:2,000, #3868, Cell Signaling Technology) at 4℃ overnight, followed by incubation with Alexa Fluor 488-conjugated secondary (#2975, Cell Signaling Technology) at room temperature for 40 min. The staining was analyzed under fluorescence microscopy.