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Total protein lysates were prepared from lung tissues of ALI mice using RIPA Buffer (89900, ThermoFisher, USA) and quantitated using a BCA protein assay kit (A53227, ThermoFisher, USA). Forty microliter of protein and 5 μl of marker (PR1910, Solarbio, China) were separately loaded on 12% SDS-PAGE gel (P0053A, Beyotime, Shanghai, China) and subjected to electrophoresis. Afterwards, the protein was transferred onto PVDF membranes (P2438, Sigma-Aldrich, USA), and the membranes were blocked by Tris Buffered Saline and 1% Tween 20 (TBST, TA-125-TT, ThermoFisher, USA) containing 5% skimmed milk. Then, the membranes were incubated at 4℃ overnight with primary antibodies against Occludin (SAB35 00301, 57 kDa, 1：1,000, Sigma-Aldrich, USA), SPC (ab2 11326, 21 kDa, 1：1,000, Abcam, Cambridge, MA, USA), FGF2 (SAB2108135, 31 kDa, 1：1,000, Sigma-Aldrich, USA), and GAPDH (ab181602, 36 kDa, 1：10,000, Abcam, USA). Then, the membranes were incubated with Goat Anti-Rabbit IgG (A32733, 1：20,000, ThermoFisher, USA) at room temperature for 1 hour. The immunoreactive bands were detected on an imaging system (iBright CL1500, A44240, ThermoFisher, USA) using an enhanced chemiluminescence reagent kit (WP20005, ThermoFisher, USA). The density of the bands was quantified by ImageJ software (version 1.52s, National Institutes of Health, Bethesda, MD, USA).

After transfection, A375 cells and A2058 cells were lysed with RIPA Buffer (89900, ThermoFisher), and total protein lysates were then quantitated by BCA kits (A53227, ThermoFisher). The protein lysates (45 μg) and marker (4 μl; PR1910, Solarbio) were loaded separately and subjected to electrophoresis with 10% SDS‐PAGE gel (P0670, Beyotime). Later, the protein was transferred onto PVDF membranes (P2438, Sigma‐Aldrich) and blocked by 5% skim milk with Tris‐Buffered Saline with 1% Tween 20 (TBST; TA‐125‐TT, ThermoFisher) for 1 h. Afterward, the membranes were incubated with primary antibodies for TUBB (#2128, 55 kDa, 1:1000, Cell Signaling Technology), and GAPDH (#4292, 37 kDa, 1:500, Cell Signaling Technology) overnight at 4°C. The membranes were again washed with TBST and added with Goat anti‐Rabbit IgG (A32731, 1:10,000, ThermoFisher). An enhanced chemiluminescence reagent kit (WP20005, ThermoFisher) was used to develop immunoblots, which were later analyzed on an imaging device (iBright CL750, ThermoFisher), and quantified by ImageJ software (1.52s version, National Institutes of Health).

Processed C666‐1 cells and HK1 cells were lysed by RIPA Buffer (89,900, ThermoFisher) blended with a Protease‐Phosphatase inhibitor (A32959, ThermoFisher). The concentration of total protein lysate was determined using the BCA kit (A53227, ThermoFisher). The total protein (45 μg) and marker (5 μl) (PR1910, Solarbio) were separately loaded, separated by 10% SDS‐PAGE gel (P0670, Beyotime), and transferred onto PVDF membranes (P2438, Sigma‐Aldrich). Then, the membranes were blocked by 5% skim milk in Tris Buffered Saline with 1% Tween 20 (TBST, TA‐125‐TT, ThermoFisher) at room temperature for 1 h. Afterwards, the membranes were incubated at 4°C overnight with primary antibodies against AKT (rabbit, ab8805, 55 kDa, 1:500, Abcam), phosphorylated (p)‐AKT (rabbit, ab38449, 56 kDa, 1:500, Abcam), PI3K (rabbit, #4292, 85 kDa, 1:1000, Cell signaling technology), p‐PI3K (rabbit, #4228, 85 kDa, 1:1000, Abcam), and GAPDH (mouse, ab8245, 36 kDa, 1:10000, Abcam). After TBST washing, the membranes were incubated with secondary antibody Goat antiRabbit IgG (A32731, 1:10000, ThermoFisher) or Goat antiMouse IgG (A32733, 1:1000, ThermoFisher). The protein bands were visualized using the enhanced chemiluminescence reagent (WP20005, ThermoFisher) and analyzed using ImageJ software (1.52 s version, National Institutes of Health). All protein expressions were normalized to GAPDH.