X omat ar x ray film

A total of 50 μg of protein from each sample was separated through SDS-PAGE then transferred to a 0.45 mm pore nitrocellulose membrane with an Enhanced ChemiLuminescence (ECL) Semidry Blotter (Amersham Biosciences, USA). The membrane was blocked with 5% skim milk in TBST (20 mM Tris-HCl, 150 mM NaCl, and 0.05% Tween-20) at 37°C for 1 h. The membranes were then incubated with polyclonal antibodies diluted to 1:400 in 5% skimmed milk at 4°C for 12 h. After washing the membrane for three times with TBST for 10 min, the membrane was subsequently incubated with diluted goat-anti mouse or goat-anti rabbit IgG (Sangon, China) diluted to 1:3000 at room temperature for 1 h. The membrane was further washed with TBST for three times and incubated in Western Lightning-ECL substrate (Perkin Elmer, USA) prior to exposure to a X-OMAT AR X-ray film (Eastman Kodak, USA). Band density was analyzed with ImageJ, and protein expression levels were normalized to that of β-Tubulin control.
All antibodies used in this study could be found in the key resources table, Table 1. STPKLRR, Hop, FliC, and Tmod antibodies were generated in accordance with a previously described method [68 (link), 69 (link)]. The Hop, FliC, and Tmod antibodies had been successfully used in our previous works [18 (link), 70 (link), 71 (link)]. The specificity of the STPKLRR antibody was shown in Fig 3A.

Mammalian and oyster mitochondria were isolated using a Cell Mitochondria Isolation Kit (Beyotime) following the manufacturer’s protocol. The mitochondrial or cytoplasmic proteins were extracted from the prepared fractions with lysis buffer. For western blotting, proteins were resolved by 12% SDS-PAGE and transferred onto a 0.45 nm pore nitrocellulose membrane with the use of Semi-dry blotter (GenScript, Jiangsu, China). The membrane was then blocked with TBST (20 mmol/l Tris-HCL, 150 mmol/l NaCl, and 0.05% Tween-20) containing 5% skimmed milk at room temperature for 1 h, and then incubated with the appropriate antibodies at 4 °C overnight, washed three times with TBST, incubated with peroxidase-labeled secondary antibodies at room temperature for 1 h, and finally washed five times with TBST. Western Lightning Plus-ECL substrate (PerkinElmer, Waltham, MA, USA) was then added to membranes, which were exposed to X-OMAT AR X-ray film (Eastman Kodak, Rochester, NY, USA).

The protein concentrations of coelomocyte lysates were determined with a BCA Protein Assay Kit (Sangon). Approximately 50 μg of protein was separated with 10% SDS-polyacrylamide gels and transferred to 0.45μm ECL membranes. After blocking with 5% skim milk in TBST (50 mmol L⁻¹ Tris-HCl, 150 mmol L⁻¹ NaCl, and 1% Tween-20) at room temperature for 120 min, the membranes were incubated with antibodies diluted at 1:500 in 5% BSA solution at 4°C overnight. The AjNLRC4 antiserum was prepared as described above, the Transferrin, dynamin, caveolin and Rab5 antiserum were were purchased from (Abcam). The membranes were incubated with HRP-labelled anti-rat or mouse IgG (1:2000) in 5% BSA solution at room temperature for 1.5 h. The membranes were incubated in Western Lightning-ECL substrate (Perkin-Elmer) prior to exposure with X-OMAT AR X-ray film (Eastman Kodak, Rochester, NY). The densities of the protein bands were quantified using the ImageJcngr software package, and the results were derived from the statistical analysis of three independent experiments.